Neurochem Res (2012) 37:1697–1706 DOI 10.1007/s11064-012-0779-0
ORIGINAL PAPER
Inhibition of Nuclear Factor Erythroid 2-Related Factor 2 Exacerbates HIV-1 gp120-Induced Oxidative and Inflammatory Response: Role in HIV Associated Neurocognitive Disorder Pichili Vijaya Bhaskar Reddy • Marisela Agudelo Venkata S. R. Atluri • Madhavan P. Nair
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Received: 16 December 2011 / Revised: 29 March 2012 / Accepted: 4 April 2012 / Published online: 25 April 2012 Ó Springer Science+Business Media, LLC 2012
Abstract The HIV epidemic continues to be the most severe public health problem and concern within USA and across the globe. In spite of the highly active antiretroviral therapy, HIV infected subjects experience major neurological complications that range from HIV associated dementia to moderate neurocognitive and motor impairments collectively termed as HIV associated neurocognitive disorders (HAND). Astrocytes play an important role in the neuropathogenesis of HAND. Further, in the recent years it has been shown that oxidative stress plays a major role in the neuropathogenesis of HAND. Nuclear factor erythroid 2-related factor 2 (Nrf2), a leucine zipper redox-sensitive transcription factor, is an important regulator of cell survival and adaptive mechanisms and has been shown to possess a protective role in a variety of neurological and inflammatory disorders. Earlier we have shown that Nrf2 is upregulated in response to HIV-1 gp120 and such upregulation of Nrf2 may be a protective mechanism against the HIV-induced oxidative stress. We hypothesize that Nrf2mediated antioxidant pathways are important in regulating the HIV-induced oxidative stress and that the disruption of Nrf2 makes the cells more susceptible to HIV gp120induced deleterious effects. Our results indicate that when astrocytes are exposed to gp120 there is an increase in the expression of NOX2, a subunit of NADPH oxidase, and also an upregulated expression of nuclear factor kappa B, tumor necrosis factor-a (TNF-a) and matrix metalloproteinase-9
P. V. B. Reddy M. Agudelo V. S. R. Atluri M. P. Nair (&) Department of Immunology, Institute of NeuroImmune Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL, USA e-mail:
[email protected] P. V. B. Reddy e-mail:
[email protected]
(MMP-9). However, the degree of expression was significantly higher in those cells where Nrf2 was silenced by siRNA. Taken together, these results suggest a possible protective role of Nrf2 in regulating the levels of pro-oxidative and pro-inflammatory molecules in HAND. Keywords Nrf2 Oxidative stress Inflammation NADPH oxidase HIV
Introduction The era of highly active antiviral therapy (HAART) has significantly controlled AIDS and disorders linked to it. However, the prevalence of HIV-associated neurocognitive disorders (HAND) is increasing as HIV infected individuals are living longer. HAND is characterized by the development of cognitive, behavioral and motor abnormalities and occurs in about 50 % of the HIV infected individuals [1–3]. Earlier studies have shown that HIV can infect and replicate in different kinds of cells in the brain such as macrophages, microglia and monocytes [1, 4]. However, astrocytes are thought to be the most important reservoir in eliciting the inflammatory responses against HIV-1 [5–7]. The viral proteins such as HIV-1gp120, Tat, vpr are known to induce the release of neurotoxic factors which lead to apoptotic cell death [8–10]. As the number of treated individuals with HIV infection is raising, the frequency of HAND is also increasing. In the recent years it has been shown that oxidative stress plays a major role in the neuropathogenesis of HAND [11–14]. Oxidative stress sets in when there is an imbalance between excessive reactive oxygen species (ROS) generated and endogenous cellular antioxidant defenses [15, 16]. Studies have demonstrated that HIV-1
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gp120 and Tat induce oxidative stress in astrocytes leading to cell death [5, 17–23]. In addition, evidences suggest that chronic oxidative stress prevails in AIDS patients and the signs and consequences of such oxidative stress are reflected in astrocytes and neurons of HAND patients [24– 26]. A combination of several factors including HIV-1 gp120, Tat and other viral mediators that are released from the glial cells are believed to induce oxidative stress and thus involved in the neuropathogenesis of HAND [1, 3]. Nuclear factor erythroid 2-related factor 2 (Nrf2), a leucine zipper redox-sensitive transcription factor, is an important regulator of cell survival and adaptive mechanisms. In normal healthy condition, Nrf2 is sequestered in the cytoplasm by a cytosolic regulatory protein, Keap1 [27–29]. However, in conditions of elevated oxidative stress Nrf2 translocates from cytoplasm to the nucleus and sequentially binds to a promoter sequence called the antioxidant response element (ARE), resulting in the expression of antioxidant and cytoprotective genes that have the potential to attenuate cellular damage. Earlier studies have shown that Nrf2 has a protective role in a variety of neurological and inflammatory disorders [30–35]. Recent studies from our laboratory and from others have shown that Nrf2 is upregulated in response to HIV Tat and gp120 treatments [36, 37]. Such upregulation of Nrf2 may be a protective mechanism against the oxidative stress in HAND. Oxidative stress and inflammation plays a major role in HAND during which NF-kappa B and TNF-a are known to initiate several pathways including MMP-9. However the link between Nrf2, oxidative stress, and inflammation in HAND has not been studied. We hypothesize that the ability of Nrf2 is important in regulating the oxidative, inflammatory cascades in HAND and that the disruption of Nrf2 makes the cells more susceptible to oxidative and inflammatory damage.
Materials and Methods Primary cultures of human astrocytes (HAs; catalog# 1800) were purchased from Sciencell Laboratories (Carlsbad, CA) and cultured as per supplier’s instructions. Astrocytes were maintained in basal medium containing 10 % fetal bovine serum, 50 units/ml penicillin, astrocytes growth supplement, and 100 lg/ml streptomycin (Sciencell, CA) at 37 °C with 5 % CO2. HIV-1 clade B recombinant protein was obtained from NIH AIDS Reagent program. Nrf2 siRNA was obtained from Santa Cruz Biotechnology. In order to perform gene expression studies, astrocytes were cultured in 6-well tissue culture plates. In an earlier study [36] we have established that 50 ng/ml gp120 treatment for 24 h was the optimal concentration for astrocytes to induce Nrf2. For the siRNA studies, the cells were
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transfected with siRNA against Nrf2 or a nonspecific control siRNA (scramble) for 24 h and then treated with gp120. Following the treatments the cells were used for either gene or protein studies. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) At the end of the treatment periods the cells were harvested and mRNA was isolated by using the RNAeasy kit obtained from Qiagen. (Qiagen, GmbH, Germany). Following the RNA extraction, cDNA was synthesized using high-capacity reverse transcriptase cDNA kit (Applied Biosystems, Foster City, CA, USA) to perform qRT-PCR using Taqman gene expression assays for NOX2, TNF-a, MMP-9 and Nrf2. GAPDH served as an internal control. Relative abundance of each mRNA was assessed using brilliant Q-PCR master mix obtained from Stratagene and by using MX 3000P instrument. Relative mRNA expression was quantitated and the mean fold change in expression of the target gene was calculated using the comparative ct method as previously described [36]. Inhibition of Nrf2 by siRNA Specific siRNA against Nrf2 (Santa Cruz Biotechnology, Cat #37030) was used in the current study. Control siRNA or scramble was obtained from the Santa Cruz Biotechnology and was used as a control (Santa Cruz Biotechnology, Cat #H2310). Transfection of siRNA in astrocytes was performed using the siRNA transfection reagent (Santa Cruz Cat#Sc-29528). For siRNA knockdown of Nrf2, astrocytes were transfected with 40 pmol siRNA for 48 h in the transfection medium containing transfection reagent according to the manufacturer’s protocol. The control siRNA used had at least four mismatches to all known human, mouse and rat genes. For the mock transfection, we used the transfection buffer and transfection reagents containing no siRNA. Western Blotting Primary astrocytes (2 9 106) were seeded and grown to 70 % confluence prior to treatments. Treated and untreated control cultures were then washed twice with ice-cold PBS and scraped directly into lysis buffer containing protease inhibitors (Pierce Chemical, Rockford, IL). Equal amounts of protein were loaded in each well and were run on SDSPAGE. The separated proteins were then electrophoretically transferred to polyvinylidene difluoride membranes at 100 V for 1 h. The membranes were blocked with 5 % nonfat milk in 3 % Tris-buffered saline containing 0.1 %
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Tween-20 for 40 min at room temperature. The membranes were then incubated with primary antibodies, anti Nrf2 (Santa Cruz biotechnology; Cat#722-c), anti NF-kappa B p65 (Cell signaling; Cat# 4764S), p50 subunit (Millipore; Cat#AB69) or anti-b-actin (Cell Signaling; Cat# 49701), NQO1 (Cell Signaling; Cat# 3187) at 4 °C overnight, followed by washing and a second incubation in HRP-conjugated secondary antibodies (1 h at RT). Antibody binding to protein on the membranes was visualized using chemiluminescence, and the optical densities of individual bands were quantitated using the Chemi-Imager digital imaging system. Preparation of Nuclear Extract and Determination of NFjB Expression At the end of the treatment periods primary astrocyte cultures were washed twice with ice-cold PBS, harvested into 1 ml of PBS and then centrifuged at 3,000 rpm for 5 min at 4 °C. The cell pellet was suspended in 200 ll of buffer containing 10 mM HEPES (pH 7.9), 10 mM Kcl, 0.1 mM EDTA, 0.1 mM EGTA, 1 lM dithiothreitol and protease inhibitor. The cell pellet obtained was incubated on ice for 15 min following which 15 ll of NP-40 was added and vortexed thoroughly. An aliquot of the each of the samples were used for protein estimation. Equal amount of protein was centrifuged at 3,000 rpm for 3 min at 4 °C. The resulting nuclear pellet was suspended in 30 ll of cold buffer containing 20 mM HEPES (pH 7.9), 0.4 mM Nacl, 1 mM EDTA, 1 mM EGTA, 1 lM dithiothreitol and protease inhibitor cocktail. The pellet was incubated on ice for 15 min with occasional vortex. The extract was then centrifuged at 10,000 RPM for 6 min. The supernatant containing nuclear extract was loaded on SDS–Polyacrylamide gel and western blots with antibodies for NFjB subunits were performed as described above. Lamin antibody that is specific for nuclear protein was used as a loading control. For determining the NFKB activity the cells were seeded in regular astrocyte medium without serum and antibiotics. Following the transfection the cells were lysed in 150 ml in a microfuge tube. The lysate was vortexed for 30 s and then the supernatants were collected by centrifuging at 10,000 rpm for 2 min. The activity in the supernatant was measured and expressed as relative activity normalizing to controls. Enzyme-Linked Immunosorbant Assay At the end of the treatment periods the protein levels of TNF-a and MMP-9 using commercially available ELISA kits as per the manufacturer’s instructions (TNF-a: Biolegend, San Diego, CA; MMP-9: Invitrogen, San Diego, CA).
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Measurement of ROS ROS levels were detected using dichlorofluorescein diacetate assay (DCF-DA; Molecular Probes, Eugene, OR). The Nrf2 transfected cells were divided into resveratrol exposed or unexposed groups. To the resveratrol group, 25 lM resveratrol was supplemented. Then the cells were treated with gp120 for 24 h. The cells were later treated with DCFDA 100 lM for 30 min. At the end of the treatment period, the fluorescence was read in a BioTek Synergy HT microplate reader (excitation 485 nm and emission 528 nm; BioTek, Winooski, VT). Statistical Analysis All experiments were conducted in duplicate wells in the samples derived from three separate and independent experiments. The data were subjected to analysis of variance followed by Tukey’s test for comparison. Data were represented as mean SEM. p \ 0.05 was considered significant.
Results Nrf2 siRNA Transfection Reduces Nrf2 Expression In order to examine the role of Nrf2, we inhibited the expression of Nrf2 using siRNA that was directed against Nrf2. Primary astrocytes were transfected with Nrf2 siRNA for 48 h and the extent of mRNA silencing as well as well as protein expression were determined. Nrf2 mRNA levels were reduced by about 70 % (p \ 0.0001) in Nrf2-siRNA transfected astrocytes (Fig. 1a). Similar to the gene expression, there was a significant inhibition in the Nrf2 protein expression as well (65 %, p \ 0.008) (Fig. 1b, c). To avoid any non-specific effects, astrocytes were transfected with a control siRNA (scramble) for 48 h. The transfection of the control siRNA (scramble) showed no changes in either the gene or protein expression levels. In order to investigate if the suppression of Nrf2 by siRNA further down regulates the downstream genes, we examined the gene and protein expression of NAD(P)H dehydrogenase quinone1(NQO1). NQO1 is a downstream gene that is regulated by Nrf2 and plays a role in oxidative stress mediated events. Our results showed that the transfection of siRNA against Nrf2 significantly reduced the NQO1gene (p \ 0.001) (Fig. 1d) as well as protein (p \ 0.01) expression (Fig. 1e, f). Inhibition of Nrf2 Results in Increased NOX-2 in Astrocytes Exposed to gp120 Nrf2 has been reported to control the redox status of the cell by regulating the levels of ROS production. In the
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Fig. 1 Effect of Nrf2 inhibition on HIV-1 gp120-induced mRNA and protein expression. Astrocytes were seeded in six well plates. On the following day, Cultures were transfected with siRNA against Nrf2 for 48 h. At the end of the transfection, cells were harvested separately for mRNA and protein analysis. Scramble indicates a group where nonspecific siRNA was transfected. a Analysis of mRNA levels by qRTPCR show a significant inhibition of Nrf2 gene in the Nrf2siRNA transfected group as compared to either control or scramble. b Western blot showing significant reduction of Nrf2 protein
expression post transfection of cultures to siRNA against Nrf2 as compared to non-transfected controls or the scramble group. c Quantification of the Nrf2 protein band intensities normalized against a house keeping gene. d Shows the suppression of the mRNA levels of NQO1, a downstream molecule of Nrf2. e Western blot showing reduction of NQO1 protein expression after transfecting astrocytes with siRNA against Nrf2. f Quantification of the NQO1 protein band intensities normalized against a house keeping gene
earlier report we have demonstrated an increase in the ROS production when astrocytes were exposed to HIV-1 gp120. To study the role of Nrf2 in HIV-1 gp120-induced oxidative stress, we compared the non-transfected control cells with the cells where Nrf2 was silenced. One major source for ROS generation is NADPH oxidase which has several subunits among which NOX2 has been shown to be important and also involved in the neuropathogenesis of HAND. Hence we looked at the mRNA levels of NOX2 in the wild type and Nrf2 silenced astrocytes with and without exposure to gp120. Our results indicate that the levels of NOX2 mRNA levels were elevated in cells that were exposed to gp120 as compared to untreated controls (p \ 0.01) (Fig. 2a). However the increase in the NOX2 mRNA was greater when Nrf2 deficient cells were exposed to gp120 (p \ 0.0002). The elevation of NOX2 mRNA due to gp120 treatment was statistically higher in Nrf2 inhibited-gp120 treated cells as compared to nontransfected-
gp120 treated cells (p \ 0.01) (Fig. 2a). In addition to the gene expression our results also showed similar results in the protein expression pattern. Treatment of non-transfected astrocytes with gp120 showed an increase in NOX2 protein expression as compared to control. However its expression in the Nrf2 transfected cells was significantly higher compared to the non-transfected gp120 treated astrocytes (Fig. 2b). The cells lacking Nrf2 by itself did not differ from the control cells. The cells that were transfected with control siRNA (scramble) behaved like normal control cells (Fig. 2).
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Inhibition of Nrf2 Increases NF-kappa B NF-kappa B is activated in response to HIV recombinant proteins as well as virus infection. However whether Nrf2 plays a role in such activation is not examined. In the present study, we examined the NF-kappa B activation in
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Fig. 2 Effect of Nrf2 inhibition on NOX2. Astrocytes were seeded in six well plates. On the following day, Cultures were transfected with siRNA against Nrf2 for 48 h following which the cells were exposed to gp120 (50 ng/ml) for 24 h. Heat inactivated gp120 treated cells were used as controls. At the end of the transfection, cells were harvested and RNA was extracted and reverse transcribed for qRTPCR using Taqman expression assays against NOX2 subunit of NADPH oxidase. A house keeping gene GAPDH was used as an internal control. a HIV-1 gp120 treatment elicited significantly enhanced the upregulation of NOX2 gene expression in Nrf2 siRNA transfected cells compared to the non-transfected cells or the nonspecific siRNA transfected scramble (scr) group. b Representative western blot showing the significantly elevated expression of NOX2 protein in gp120 treated as well as Nrf2 transfected-gp120 treated cultures. The data expressed are Mean ± SD of Transcription accumulation Index (TAI) values of three independent experiments. Statistical significance was performed by ANOVA
non-transfected control as well as Nrf2-siRNA transfected astrocytes. Our results demonstrate that when treated with gp120, all the groups including non-transfected control cells, Nrf2-siRNA transfected cells and control siRNA transfected cells (scramble) showed higher levels of total p65 subunit of NFkB. In addition, the nuclear translocated p65 and p50 subunits of NFkB were significantly elevated (Fig. 3a). However, the upregulation of the NFKB p65 (total), p65 (nuclear) and p50 (nuclear) subunits in response to gp120 exposure was significantly higher in the Nrf2-siRNA transfected-gp120 exposed cells as compared to the non-transfected-gp120 treated astrocytes (Fig. 3 a). Consistent with these results, NFkB activity also showed a similar pattern. The activity was significantly higher in the siRNA transfected gp120 treated group compared to the non-transfected gp120 treated cells (Fig. 3b).
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Fig. 3 Influence of Nrf2 inhibition on the protein expression of NFkappa B. Astrocytes were seeded in six well plates. On the following day, Cultures were transfected with siRNA against Nrf2 for 48 h following which the cells were exposed to gp120 (50 ng/ml) for 24 h. At the end of the transfection, cells were harvested and protein was subjected to western blot analysis. a The figure shows a representative blot demonstrating a differential expression of p65 and p50 subunits of NF-kappa B protein in total cell lysate as well as in the nuclear fractions isolated from the cells that contain Nrf2 against those that lack Nrf2. A house keeping gene actin was used to normalization in the total cell lysate while lamin was used for normalization in the nuclear fractions. HIV-1 gp120 treatment elicited significantly enhanced the upregulation of NF-kappa B expression in Nrf2 siRNA transfected cells as compared to the non-transfected cells or the nonspecific siRNA transfected scramble (scr) group. b Represents the relative NF kappa B activity
Inhibition of Nrf2 Results in Increased TNF-a in Astrocytes Exposed to gp120 TNF-a has been shown to play a role in the neuropathogenesis of HAND. However the role of Nrf2 in TNF-a regulation has not been studied. To determine the role of Nrf2 in inflammation, we examined the levels of TNF-a in control as well as the Nrf2 siRNA transfected astrocytes. Non-transfected astrocytes exposed to gp120 showed increase in the mRNA levels of TNF-a as measured by the qRTPCR (p \ 0.01). In contrast to the non-transfected astrocytes, the Nrf2-siRNA transfected cells were more sensitive to gp120 treatment and demonstrated higher levels of TNF-a (p \ 0.01) (Fig. 4). The Nrf2-siRNA transfected astrocytes did not show any significant
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Fig. 4 Effect of Nrf2 inhibition on TNF-a expression. Cultures were transfected with siRNA against Nrf2 for 48 h following which the cells were exposed to gp120 (50 ng/ml) for 24 h. Heat inactivated gp120 treated cells were used as controls. a HIV-1 gp120 treatment elicited significantly higher levels of TNF-a in the Nrf2 siRNA transfected cells as compared to the non-transfected cells or the nonspecific siRNA transfected scramble (scr) group. b The supernatants were collected and analyzed for TNF-a protein levels by ELISA. The data expressed are Mean ± SD of Transcription accumulation Index (TAI) values of three independent experiments. Statistical significance was performed by ANOVA
alterations by itself, however when treated with gp120 showed an increased TNF-a expression that was much higher than the non-transfected-gp120 treated cells (p \ 0.01). This difference was significantly higher when compared to the gp120 treatment in normal non-transfected astrocytes (p \ 0.0003). Transfection of the control siRNA (scramble) by itself did not alter the levels of TNF-a and when treated with gp120 showed similar increase as that of the gp120 treatment in non-transfected cells (p \ 0.01) (Fig. 4a). Data presented in fig. 4b show the effect of gp120 on the TNF-a protein secretion in the non-transfected control cells (13.5 ± 7.6 pg/ml), non-transfectedgp120-treated cells (52.68 ± 14.01 pg/ml), Nrf2 transfected-gp120 treated cells (112.9 ± 25.65 pg/ml) and control siRNA transfected-gp120 treated (44.3 ± 5.09 pg/ml). Similar to the gene expression the protein secretion was higher in the Nrf2 transfected-gp120 treated cells as compared to the non-transfected gp120 treated cells. The
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Fig. 5 Influence of Nrf2 inhibition on MMP-9. Astrocytes were seed in six well plates. On the following day, Cultures were transfected with siRNA against Nrf2 for 48 h following which the cells were exposed to gp120 (50 ng/ml) for 24 h. a HIV-1 gp120 treatment elicited significantly higher levels of MMP-9 mRNA expression in the Nrf2 siRNA transfected cells as compared to the non-transfected cells or the nonspecific siRNA transfected scramble (scr) group. b The supernatants were collected and analyzed for MMP-9 protein levels by ELISA. The data expressed are Mean ± SD of Transcription accumulation Index (TAI) values of three independent experiments. Statistical significance was performed by ANOVA
TNF-a secretion in the control siRNA transfected cells was similar to that of the control cells (Fig. 4b). Inhibition of Nrf2 Exacerbates gp120-Induced Expression of MMP-9 in Astrocytes Since we found that TNF-a levels are exacerbated in Nrf2 inhibited astrocytes, our aim was to examine the effects on MMP-9 since increased TNF-a production has been shown to upregulate MMP-9. Previous studies have demonstrated that HIV-virus as well as recombinant proteins result in the increased expression of MMP-9. How Nrf2 regulates MMP-9 has not been examined. Here we compared the MMP-9 levels in non-transfected astrocytes versus those cells where Nrf2 was silenced using specific siRNA. Our results show that the treatment of astrocytes with gp120 resulted in enhanced MMP-9 gene expression as compared to the control cells (p \ 0.009) (Fig. 5a). When Nrf2 silenced astrocytes were exposed to gp120, MMP-9 expression was
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significantly elevated even when compared to the nontransfected-gp120 treated cells (p \ 0.02). MMP-9 expression in the control Nrf2-siRNA transfected cells was in the similar range as that of the non-transfected control cells (p \ 0.009) (Fig. 5a). Data presented in fig. 5b show the effect of gp120 on the MMP-9 protein secretion in the non-transfected control cells (56.89 ± 13.07 pg/ml), nontransfected-gp120-treated cells (183 ± 17.7 pg/ml) Nrf2 transfected-gp120 treated cells (369.6 ± 31.22 pg/ml) and control siRNA transfected-gp120 treated (179.02 ± 20.08 pg/ml). The protein secretion was higher in the Nrf2 transfected-gp120 treated cells as compared to the nontransfected gp120 treated cells. The MMP-9 secretion in the control siRNA transfected cells was in the range of control cells (Fig. 5b). Activation of Nrf2 Suppresses HIV-gp120 Induced ROS Earlier studies have shown that exposure of astrocytes to HIV virus or its recombinant proteins results in increased production of ROS. It has also been reported that the increased ROS production leads to oxidative stress and also initiates the inflammatory response. In order to examine the protective role of Nrf2 in HIV-1 induced oxidative damage we measured the levels of ROS in the cells that contain Nrf2 versus those cells that lack Nrf2. In order to examine this aspect we used resveratrol which is known to induce Nrf2. Figure 6a shows that resveratrol significantly upregulates Nrf2. We further examined if the HIV-induced ROS production is reduced after the treatment with the small molecule Nrf2 activator resveratrol. Our results suggest that the cells that are deficient in Nrf2 show increased ROS generation as compared to the control cells. However an upregulation of Nrf2 significantly reduced the HIV-induced ROS production. These results together suggest that the Nrf2 upregulation may play a protective role in HIV.
Discussion With the developments in HAART, there has been a significant improvement in the immune outcome and survival rate of the patients infected with HIV [1, 3]. However neurological abnormalities still persist in these patients. HAND is manifested by enhanced neuroinflammation, reactive astrocytosis, formation of multinucleated giant cells, blood brain barrier damage, formation of microglial nodules and neural apoptosis associated with increased viral replication and deterioration of the immune responses [38–40]. Based on the severity of the neurocognitive dysfunction, HAND has been classified into 3 major calluses
Fig. 6 Astrocytes were seeded in six well plates. Cultures were transfected with siRNA against Nrf2 for 48 h. Cultures were pretreated with resveratrol before exposure to gp120. a Resveratrol (25 lM) induces the upregulation of Nrf2. b DCF-DA was added one hour prior to the gp120 treatment. Levels of ROS were measured with Biotek synergy HT microplate reader. The fluorescence was detected at 485 excitation and 528 emission wave lengths. The data expressed are Mean ± SD of Transcription accumulation Index (TAI) values of three independent experiments. Statistical significance was performed by ANOVA
namely asymptotic neurocognitive impairment, minor neurocognitive disorder and the most severe HIV associated dementia [3]. Oxidative stress in the CNS is implicated in the development of neurological disorder associated with HIV infection. Earlier studies have reported that HIV-1 viral proteins including gp120 and tat released from the infected cells induce oxidative stress in the CNS either directly and indirectly [11, 26, 41–43]. Increased ROS production in CNS cells has been demonstrated to play a major role in initiating several signaling cascades in HAND. Consistent with these reports, our previous results have also demonstrated an increased ROS generation by gp120 exposure to astrocytes in vitro [36]. Elevated ROS generation disturbs the redox state within the cell and has been shown to affect the immune responses and alter the cytokine expression [44, 45]. Therefore it is important to delineate the pathways that control the cellular redox homeostasis and is vital in understanding the neuropathogenesis of HAND. In order to prevent a cell from oxidative stress mediated cascades, stimulation of the antioxidant genes are critical. Nrf2 regulates the antioxidant defensive system and acts as a modifier of oxidative stress by binding to the antioxidant
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responsive element (ARE) located in the promoter regions of antioxidant genes. Stimulation of antioxidant defenses via Nrf2 upregulation has been reported to suppress the oxidative damage in sepsis and neurological disease models [46, 47]. However, the link between Nrf2, oxidative stress, inflammation and their role in the neuropathogenesis of HAND has not been explored. Although the molecular mechanisms underlying HIV-1 gp120-induced ROS production in astrocytes are not yet completely understood, NADPH oxidase is one of the potential cellular sources of ROS generation. NADPH oxidase is present in various types of cells, including monocytes/macrophages, endothelial cells, and astrocytes [48]. Recent studies revealed that NOX2 subunit of NADPH oxidase is involved in HIV-1-mediated ROS generation [5, 49, 50]. Inhibition of NADPH oxidase with Nox2 knockdown and pharmacological inhibitors, diphenyleneiodonium (DPI) and apocynin, significantly attenuated HIV-1 Tat-induced production of inflammatory mediators such as TNF-a, IL-6, and MCP-1 in microglia and macrophages [41, 51]. Activation of NADPH oxidase in neurons can contribute to cell death under stress stimuli. In the present study we report for the first time that that the astrocytes that are deficient in Nrf2 show increased NOX2 expression as compared to the untreated controls or gp120 treated cells. Consistent with these results Kong et al. [52] have reported that increased polymicrobial sepsis and LPS shock cause a higher mortality in mice models where Nrf2 was knocked out. NF-jappa B is an important element in the biological response to stress. Normally, NF-jappa B is activated by physiological as well as pathological stimuli. Activation of NF-kappa B signaling has been shown to play an important role in inducing oxidative stress and increased inflammatory response mediated by HAND [53, 54]. NF-kappa B can also be activated by induction of oxidative stress [54]. Upregulation of NF-kappa B results in stimulating several inflammatory genes that play a vital role in HAND [55]. Further deregulation of the inflammatory molecules has been well established in HAND [17, 50]. Earlier studies have shown connection between the Nrf2 and NF-kappa B mediated inflammatory cascades [56, 57]. Our results demonstrate that cells that lack Nrf2 show increased NF-kappa B activation as compared to wild type cells in response to gp120 exposure. Such increase in NF-kappa B activity has been reported to cause increased expression of inflammatory molecules [53, 55]. Consistent with our results it has been shown that the Nrf2 deficient mice exhibited higher expression of proinflammatory responses in several conditions such as sepsis [56], emphysema, traumatic brain injury [58]. Further, Nrf2 has been demonstrated to suppress the generation of ROS mediated NF-kappa B activation as well as inflammation [59, 60].
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One prime molecule that has been shown to initiate the inflammation and is released in the early phases of inflammation is TNF-a. When treated with gp120, our results showed TNF-a expression in the cells lacking Nrf2 is significantly higher as compared to the gp120 treatment of wild type cells with intact Nrf2. Increased TNF-a has been reported to promote the vascular permeability across the blood brain barrier. Further, TNF-a is also known to induce the synthesis of matrix metalloproteinase MMP-9 [58, 61]. MMPs are a class of endopeptidases that are known to damage the BBB by destroying the extracellular matrix proteins. Earlier studies have shown upregulation of MMP-9 in astrocytes treated with HIV-1 virions and its proteins such as gp120 and Tat [62, 63]. MMP-9 has been reported to be increased in the CSF of HIV infected neurologically impaired patients as well [64]. Further, the increased expression of MMP-9 via TNF-a upregulation has been earlier reported [62]. In addition, increased expression of NF-kappa B has been demonstrated to be mediated by the Nrf2 pathway thereby resulting in increased MMP-9 expression [58]. The expressions of the MMPs have been further shown to be regulated by NF-jappa B [65]. Overall, our study demonstrates that the inhibition of Nrf2 by siRNA in astrocytes showed a significant increase in NOX-2, NF-kappa-B, TNF-a, and MMP-9 levels as compared to the untreated controls or those treated with HIV-1 gp120 viral protein or a negative control siRNA. Our results suggest that the loss of Nrf2 may lead to increased susceptibility of the cells to HIV-1-induced oxidative and inflammatory damage. Together, these results suggest that the Nrf2 might play a protective role in HIV associated neurocognitive disorder. These findings suggest that the Nrf2 may be beneficial in preventing the oxidative stress and inflammatory cascades induced in HAND and may provide insight in the development of novel therapeutic strategies against HAND. Acknowledgments This study was supported by the grants from National Institute of Health (NIH) RO1DA021537, R37DA025576, and RO1MH085259.
References 1. Ellis R, Langford D, Masliah E (2007) HIV and antiretroviral therapy in the brain: neuronal injury and repair. Nat Rev Neurosci 8:33–44 2. Woods SP, Moore DJ, Weber E, Grant I (2009) Cognitive neuropsychology of HIV-associated neurocognitive disorders. Neuropsychol Rev 19:152–168 3. McArthur JC, Brew BJ (2010) HIV-associated neurocognitive disorders: is there a hidden epidemic? Aids 24:1367–1370 4. Nath A, Geiger J (1998) Neurobiological aspects of human immunodeficiency virus infection: neurotoxic mechanisms. Prog Neurobiol 54:19–33
Neurochem Res (2012) 37:1697–1706 5. Yang Y, Yao H, Lu Y, Wang C, Buch S (2010) Cocaine potentiates astrocyte toxicity mediated by human immunodeficiency virus (HIV-1) protein gp120. PLoS ONE 5:e13427 6. Minagar A, Shapshak P, Fujimura R, Ownby R, Heyes M, Eisdorfer C (2002) The role of macrophage/microglia and astrocytes in the pathogenesis of three neurologic disorders: HIVassociated dementia, Alzheimer disease, and multiple sclerosis. J Neurol Sci 202:13–23 7. Thompson KA, McArthur JC, Wesselingh SL (2001) Correlation between neurological progression and astrocyte apoptosis in HIV-associated dementia. Ann Neurol 49:745–752 8. Lipton SA (1992) Models of neuronal injury in AIDS: another role for the NMDA receptor? Trends Neurosci 15:75–79 9. Ushijima H, Nishio O, Klocking R, Perovic S, Muller WE (1995) Exposure to gp120 of HIV-1 induces an increased release of arachidonic acid in rat primary neuronal cell culture followed by NMDA receptor-mediated neurotoxicity. Eur J Neurosci 7: 1353–1359 10. Ilyin SE, Plata-Salaman CR (1997) HIV-1 gp120 modulates hypothalamic cytokine mRNAs in vivo: implications to cytokine feedback systems. Biochem Biophys Res Commun 231:514–518 11. Aksenov MY, Hasselrot U, Bansal AK, Wu G, Nath A, Anderson C, Mactutus CF, Booze RM (2001) Oxidative damage induced by the injection of HIV-1 Tat protein in the rat striatum. Neurosci Lett 305:5–8 12. Aksenov MY, Hasselrot U, Wu G, Nath A, Anderson C, Mactutus CF, Booze RM (2003) Temporal relationships between HIV-1 Tat-induced neuronal degeneration, OX-42 immunoreactivity, reactive astrocytosis, and protein oxidation in the rat striatum. Brain Res 987:1–9 13. Bansal AK, Mactutus CF, Nath A, Maragos W, Hauser KF, Booze RM (2000) Neurotoxicity of HIV-1 proteins gp120 and Tat in the rat striatum. Brain Res 879:42–49 14. Sacktor N, Haughey N, Cutler R, Tamara A, Turchan J, Pardo C, Vargas D, Nath A (2004) Novel markers of oxidative stress in actively progressive HIV dementia. J Neuroimmunol 157: 176–184 15. Betteridge DJ (2000) What is oxidative stress? Metabolism 49:3–8 16. Reddy NM, Kleeberger SR, Kensler TW, Yamamoto M, Hassoun PM, Reddy SP (2009) Disruption of Nrf2 impairs the resolution of hyperoxia-induced acute lung injury and inflammation in mice. J Immunol 182:7264–7271 17. Louboutin JP, Reyes BA, Agrawal L, Van Bockstaele EJ, Strayer DS (2010) HIV-1 gp120-induced neuroinflammation: relationship to neuron loss and protection by rSV40-delivered antioxidant enzymes. Exp Neurol 221:231–245 18. Ronaldson PT, Bendayan R (2008) HIV-1 viral envelope glycoprotein gp120 produces oxidative stress and regulates the functional expression of multidrug resistance protein-1 (Mrp1) in glial cells. J Neurochem 106:1298–1313 19. Blokhina O, Virolainen E, Fagerstedt KV (2003) Antioxidants, oxidative damage and oxygen deprivation stress: a review. Ann Bot 91:179–194 20. Nath A (2002) Human immunodeficiency virus (HIV) proteins in neuropathogenesis of HIV dementia. J Infect Dis 186(Suppl 2):S193–S198 21. Pocernich CB, La Fontaine M, Butterfield DA (2000) In vivo glutathione elevation protects against hydroxyl free radicalinduced protein oxidation in rat brain. Neurochem Int 36: 185–191 22. Pocernich CB, Poon HF, Boyd-Kimball D, Lynn BC, Nath A, Klein JB, Butterfield DA (2005) Proteomic analysis of oxidatively modified proteins induced by the mitochondrial toxin 3-nitropropionic acid in human astrocytes expressing the HIV protein tat. Brain Res Mol Brain Res 133:299–306
1705 23. Viviani B, Corsini E, Binaglia M, Galli CL, Marinovich M (2001) Reactive oxygen species generated by glia are responsible for neuron death induced by human immunodeficiency virus-glycoprotein 120 in vitro. Neuroscience 107:51–58 24. Chauhan A, Turchan J, Pocernich C, Bruce-Keller A, Roth S, Butterfield DA, Major EO, Nath A (2003) Intracellular human immunodeficiency virus Tat expression in astrocytes promotes astrocyte survival but induces potent neurotoxicity at distant sites via axonal transport. J Biol Chem 278:13512–13519 25. Mattson MP, Haughey NJ, Nath A (2005) Cell death in HIV dementia. Cell Death Differ 12(Suppl 1):893–904 26. Turchan J, Pocernich CB, Gairola C, Chauhan A, Schifitto G, Butterfield DA, Buch S, Narayan O, Sinai A, Geiger J et al (2003) Oxidative stress in HIV demented patients and protection ex vivo with novel antioxidants. Neurology 60:307–314 27. Ma Q, Kinneer K, Bi Y, Chan JY, Kan YW (2004) Induction of murine NAD(P)H:quinone oxidoreductase by 2,3,7,8-tetrachlorodibenzo-p-dioxin requires the CNC (cap ‘n’ collar) basic leucine zipper transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2): cross-interaction between AhR (aryl hydrocarbon receptor) and Nrf2 signal transduction. Biochem J 377:205–213 28. Motohashi H, Yamamoto M (2004) Nrf2-Keap1 defines a physiologically important stress response mechanism. Trends Mol Med 10:549–557 29. Nguyen T, Sherratt PJ, Nioi P, Yang CS, Pickett CB (2005) Nrf2 controls constitutive and inducible expression of ARE-driven genes through a dynamic pathway involving nucleocytoplasmic shuttling by Keap1. J Biol Chem 280:32485–32492 30. Alfieri A, Srivastava S, Siow RC, Modo M, Fraser PA, Mann GE (2011) Targeting the Nrf2-Keap1 antioxidant defence pathway for neurovascular protection in stroke. J Physiol 589:4125–4136 31. Chen G, Fang Q, Zhang J, Zhou D, Wang Z (2011) Role of the Nrf2-ARE pathway in early brain injury after experimental subarachnoid hemorrhage. J Neurosci Res 89:515–523 32. Guerrero-Beltran CE, Calderon-Oliver M, Pedraza-Chaverri J, Chirino YI (2010) Protective effect of sulforaphane against oxidative stress: recent advances. Exp Toxicol Pathol. doi:10.1016/ j.etp.2010.11.005 33. Kuang X, Scofield VL, Yan M, Stoica G, Liu N, Wong PK (2009) Attenuation of oxidative stress, inflammation and apoptosis by minocycline prevents retrovirus-induced neurodegeneration in mice. Brain Res 1286:174–184 34. Rojo AI, Innamorato NG, Martin-Moreno AM, De Ceballos ML, Yamamoto M, Cuadrado A (2010) Nrf2 regulates microglial dynamics and neuroinflammation in experimental Parkinson’s disease. Glia 58:588–598 35. Siebert A, Desai V, Chandrasekaran K, Fiskum G, Jafri MS (2009) Nrf2 activators provide neuroprotection against 6-hydroxydopamine toxicity in rat organotypic nigrostriatal cocultures. J Neurosci Res 87:1659–1669 36. Reddy PV, Gandhi N, Samikkannu T, Saiyed Z, Agudelo M, Yndart A, Khatavkar P, Nair MP (2011) HIV-1 gp120 induces antioxidant response element-mediated expression in primary astrocytes: Role in HIV associated neurocognitive disorder. Neurochem Int. doi:10.1016/j.neuint.2011.06.011 37. Zhang HS, Li HY, Zhou Y, Wu MR, Zhou HS (2009) Nrf2 is involved in inhibiting Tat-induced HIV-1 long terminal repeat transactivation. Free Radic Biol Med 47:261–268 38. Ikezu T (2009) The aging of human-immunodeficiency-virusassociated neurocognitive disorders. J Neuroimmune Pharmacol 4:161–162 39. Kanmogne GD, Schall K, Leibhart J, Knipe B, Gendelman HE, Persidsky Y (2007) HIV-1 gp120 compromises blood-brain barrier integrity and enhances monocyte migration across bloodbrain barrier: implication for viral neuropathogenesis. J Cereb Blood Flow Metab 27:123–134
123
1706 40. Toborek M, Lee YW, Flora G, Pu H, Andras IE, Wylegala E, Hennig B, Nath A (2005) Mechanisms of the blood-brain barrier disruption in HIV-1 infection. Cell Mol Neurobiol 25:181–199 41. Mollace V, Nottet HS, Clayette P, Turco MC, Muscoli C, Salvemini D, Perno CF (2001) Oxidative stress and neuroAIDS: triggers, modulators and novel antioxidants. Trends Neurosci 24:411–416 42. Nicolini A, Ajmone-Cat MA, Bernardo A, Levi G, Minghetti L (2001) Human immunodeficiency virus type-1 Tat protein induces nuclear factor (NF)-kappaB activation and oxidative stress in microglial cultures by independent mechanisms. J Neurochem 79:713–716 43. Rappaport J, Joseph J, Croul S, Alexander G, Del Valle L, Amini S, Khalili K (1999) Molecular pathway involved in HIV1-induced CNS pathology: role of viral regulatory protein. Tat J Leukoc Biol 65:458–465 44. Park J, Choi K, Jeong E, Kwon D, Benveniste EN, Choi C (2004) Reactive oxygen species mediate chloroquine-induced expression of chemokines by human astroglial cells. Glia 47:9–20 45. Song HY, Ju SM, Seo WY, Goh AR, Lee JK, Bae YS, Choi SY, Park J (2011) Nox2-based NADPH oxidase mediates HIV-1 Tatinduced up-regulation of VCAM-1/ICAM-1 and subsequent monocyte adhesion in human astrocytes. Free Radic Biol Med 50:576–584 46. Shih AY, Johnson DA, Wong G, Kraft AD, Jiang L, Erb H, Johnson JA, Murphy TH (2003) Coordinate regulation of glutathione biosynthesis and release by Nrf2-expressing glia potently protects neurons from oxidative stress. J Neurosci 23:3394–3406 47. Qiang W, Cahill JM, Liu J, Kuang X, Liu N, Scofield VL, Voorhees JR, Reid AJ, Yan M, Lynn WS, Wong PK (2004) Activation of transcription factor Nrf-2 and its downstream targets in response to moloney murine leukemia virus ts1-induced thiol depletion and oxidative stress in astrocytes. J Virol 78: 11926–11938 48. Abramov AY, Jacobson J, Wientjes F, Hothersall J, Canevari L, Duchen MR (2005) Expression and modulation of an NADPH oxidase in mammalian astrocytes. J Neurosci 25:9176–9184 49. Wu RF, Ma Z, Myers DP, Terada LS (2007) HIV-1 Tat activates dual Nox pathways leading to independent activation of ERK and JNK MAP kinases. J Biol Chem 282:37412–37419 50. Williams R, Yao H, Peng F, Yang Y, Bethel-Brown C, Buch S (2010) Cooperative induction of CXCL10 involves NADPH oxidase: Implications for HIV dementia. Glia 58:611–621 51. Turchan-Cholewo J, Dimayuga VM, Gupta S, Gorospe RM, Keller JN, Bruce-Keller AJ (2009) NADPH oxidase drives cytokine and neurotoxin release from microglia and macrophages in response to HIV-Tat. Antioxid Redox Signal 11:193–204 52. Kong X, Thimmulappa R, Kombairaju P, Biswal S (2010) NADPH oxidase-dependent reactive oxygen species mediate amplified TLR4 signaling and sepsis-induced mortality in Nrf2deficient mice. J Immunol 185:569–577 53. Shah A, Kumar A (2010) HIV-1 gp120-mediated increases in IL-8 production in astrocytes are mediated through the
123
Neurochem Res (2012) 37:1697–1706
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
NF-kappaB pathway and can be silenced by gp120-specific siRNA. J Neuroinflammation 7:96 Saha RN, Pahan K (2007) Differential regulation of Mn-superoxide dismutase in neurons and astroglia by HIV-1 gp120: implications for HIV-associated dementia. Free Radic Biol Med 42:1866–1878 Williams R, Dhillon NK, Hegde ST, Yao H, Peng F, Callen S, Chebloune Y, Davis RL, Buch SJ (2009) Proinflammatory cytokines and HIV-1 synergistically enhance CXCL10 expression in human astrocytes. Glia 57:734–743 Thimmulappa RK, Lee H, Rangasamy T, Reddy SP, Yamamoto M, Kensler TW, Biswal S (2006) Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis. J Clin Invest 116:984–995 Surh YJ, Na HK (2008) NF-kappaB and Nrf2 as prime molecular targets for chemoprevention and cytoprotection with antiinflammatory and antioxidant phytochemicals. Genes Nutr 2: 313–317 Mao L, Wang H, Qiao L, Wang X (2011) Disruption of Nrf2 enhances the upregulation of nuclear factor-kappaB activity, tumor necrosis factor-alpha, and matrix metalloproteinase-9 after spinal cord injury in mice. Mediators Inflamm 2010:238321 Chen XL, Kunsch C (2004) Induction of cytoprotective genes through Nrf2/antioxidant response element pathway: a new therapeutic approach for the treatment of inflammatory diseases. Curr Pharm Des 10:879–891 Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, Oyake T, Hayashi N, Satoh K, Hatayama I et al (1997) An Nrf2/ small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun 236:313–322 Mori N, Sato H, Hayashibara T, Senba M, Geleziunas R, Wada A, Hirayama T, Yamamoto N (2003) Helicobacter pylori induces matrix metalloproteinase-9 through activation of nuclear factor kappaB. Gastroenterology 124:983–992 Ju SM, Song HY, Lee JA, Lee SJ, Choi SY, Park J (2009) Extracellular HIV-1 Tat up-regulates expression of matrix metalloproteinase-9 via a MAPK-NF-kappaB dependent pathway in human astrocytes. Exp Mol Med 41:86–93 Moi P, Chan K, Asunis I, Cao A, Kan YW (1994) Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region. Proc Natl Acad Sci USA 91:9926–9930 Sporer B, Paul R, Koedel U, Grimm R, Wick M, Goebel FD, Pfister HW (1998) Presence of matrix metalloproteinase-9 activity in the cerebrospinal fluid of human immunodeficiency virus-infected patients. J Infect Dis 178:854–857 Bond M, Fabunmi RP, Baker AH, Newby AC (1998) Synergistic upregulation of metalloproteinase-9 by growth factors and inflammatory cytokines: an absolute requirement for transcription factor NF-kappa B. FEBS Lett 435:29–34
