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. Author manuscript; available in PMC: 2012 May 6.
Published in final edited form as: FEBS Lett. 2011 Apr 16;585(9):1375–1381. doi: 10.1016/j.febslet.2011.04.023

Novel Role of RGS2 in Regulation of Antioxidant Homeostasis in Neuronal Cells

Samina Salim 1,*, Mohammad Asghar 1, Manish Taneja 1, Iiris Hovatta 2, Yuh-Lin Wu 3, Kaustuv Saha 1, Nada Sarraj 1, Brian Hite 1
PMCID: PMC3108258  NIHMSID: NIHMS292580  PMID: 21510939

Abstract

Regulator of G-protein Signaling Protein (RGS)-2 is a modulator of anxiety and dysregulation of oxidative stress is implicated in anxiety. Also, RGS2 expression is reported to be induced by oxidative stress. Thus, if oxidative stress induces RGS2 expression and lack of RGS2 causes anxiety, then mechanisms that link RGS2 and oxidative stress potentially critical to anxiety must be revealed. Our study is the first to suggest role of RGS2 in regulation of enzymes involved in antioxidant defense namely glyoxalase-1 and glutathione reductase-1 via activation of p38 MAPK and PKC pathways in an Sp-1 dependent manner.

Keywords: Oxidative stress, Anxiety, RGS2

1. INTRODUCTION

We recently reported that increasing oxidative stress in the brain by pharmacological intervention [L-Buthionine-(S,R)-sulfoximine (BSO) treatment], increased anxiety-like behavior of rats. Interestingly, treatment with the antioxidant, tempol, reduced oxidative stress and attenuated anxiety-like behavior of rats [1]. These findings were replicated with another oxidative stress inducer, xanthine and xanthine oxidase [2]. Quite recently, two enzymes, glyoxalase (GLO)-1 and glutathione reductase (GSR)-1 involved in antioxidant defense were implicated to play an important role in anxiety [3]. Overall, the concept of involvement of oxidative stress in anxiety disorders is achieving consensus [4,5]. Interestingly, Regulator of G-protein Signaling Protein (RGS)-2 gene is associated with anxiety in animal models and in humans. RGS2 knockout mice are reported to be anxious [6,7]. There also is evidence for a linkage between RGS2 and anxiety disorder proneness and association of polymorphisms in RGS2 with panic disorder in humans [8] as well as an association between polymorphisms in RGS2 gene and anxiety-related temperament, personality, and processing of emotional information in the brain [9]. Moreover, RGS2, classically known to regulate G-protein signaling [10], is also reported to be induced by acute oxidative stress in cell culture [11]. Therefore, if oxidative stress induces RGS2 expression and lack of RGS2 causes anxiety, then mechanisms that link RGS2 and oxidative stress must be revealed. This might shed new light on mechanisms potentially relevant to the pathophysiology of anxiety disorders.

Utilizing a neuronal cell line derived from locus coeruleus (LC), we investigated regulation of RGS2, GSR1 and GLO1 in response to oxidative stress by focusing on two pathways, Protein Kinase C (PKC)and p38 MAPK. Our logic for focusing on these pathways is the following. First, oxidative stress is reported to activate PKC [12]. Second, RGS2 is reported to be induced by acute oxidative stress [11]. Third, is the reported involvement of p38 pathway in regulation of RGS2 mRNA expression [13,14]. Thus role of these two pathways in regulation of enzymes involved in antioxidant defense namely GLO1 and GSR1 via an RGS2 dependent mechanism were examined in CATH.a cells.

2. MATERIAL AND METHODS

2.1. Material

The following were purchased from the indicated sources: Hydrogen Peroxide (H2O2): Sigma Chemical Co., St. Louis, MO; fetal bovine serum and penicillin-streptomycin: Atlanta Biological, Norcross, GA; RNeasy Mini kit cat# 74104: Qiagen; Superscript one step RT-PCR kit cat# 11922–028: Invitrogen life technologies; Ethidium bromide cat# H5041, molecular ruler cat# 170–8202 and SDS-PAGE marker cat# 161–0324: Biorad. Anti-Sp-1, P-p38, p38 and RGS2 and horse-radish peroxidase conjugated anti-rabbit secondary antibodies: Santa Cruz Biotechnology Inc. Santa Cruz, CA. CATH.a cells #CRL-11179™: ATCC, VA.

2.2. Malondialdehyde assay (MDA) and protein carbonylation

CATH.a cells were treated with vehicle/ H2O2/BSO, processed and quantified for MDA as described [2,15]. Protein carbonylation was conducted using OxiSelect Protein Carbonyl ELISA kit (#STA-310, Cell Biolabs Inc. USA) per manufacturer's instructions.

2.3. Western blotting

Cells were washed with PBS, lysed, protein concentration determined in lysates [16] and subjected to western blotting [17].

2.4. PKC Activity

PKC activity was measured according to manufacturer's protocol (catalog#V5330; Promega Corporation, Madison, WI) as previously published by us [18].

2.5. Semiquantitative and quantitative (Q) RT-PCR

Total RNA was isolated from CATH.a cells after vehicle/H2O2 or BSO treatment and semiquantitative RT-PCR carried out as previously published [17] using oligonucleotide primers specific for each RGS [19]. Q-RT-PCR measurements were done on a 7300 Real Time PCR system (Applied Biosytems) using TaqManR one-step RT-PCR kit (#4309169) with pre-designed TaqMan forward and reverse primers obtained from Applied Biosystems Foster City, CA, for mouse RGS2 (ID# Mm00501385_m1), RGS4 (ID#Mm00501389_m1), GLO1 (ID#Mm00844954_s1), GSR1 (ID#Mm00833903_m1) and GAPDH primers (ID# Mm99999915_g1). Standard curves were run with serial dilutions of RNA and the PCR efficiencies determined for all oligo sets.

2.6. Transient transfection

For antisense experiments, CATH.a cells (50–60% confluent) were transiently transfected using FuGENE6 reagent (Invitrogen) with random (5'-GCCTTATTTACTACTTTCGC-3') or RGS2 antisense (5'-GCTGAATTCAAGGTC-3') oilgonucleotides. 48h later, cells were harvested and subjected to Q-RTPCR or western blotting. For luciferase assays, CATH.a cells (~60% confluent) were transiently transfected using effectene reagent with wild type or mutant RGS2 promoter luciferase construct (1.0 μg DNA/well) as well as renilla construct (0.1 μg). Renilla is a control for transfection efficiency. 24h later, the cells were treated with H2O2/vehicle (100 μM) or BSO/vehicle (1 mM) for 1h. The treatment was terminated by addition of phosphate-buffered saline. The cells were then harvested (Promega #E1910) and lysates used for luciferase assay (Promega #E1910) and bioluminescence measured on Wallac Victor2 1420 Counter. Luciferase luminescence was normalized to renilla luminescence and the ratio thus obtained was normalized to total protein.

2.7. Immunofluorescence

CATH.a cells were grown on poly D-lysine coated glass cover slips to 40–70 % confluence and exposed to vehicle/H2O2 (100 μM) for 1h and processed as previously published by us [17]. The cells were incubated with anti-Sp-1 or anti-RGS2 antibody overnight at 4°C, followed by cy3-conjugated secondary antibody for 1h in dark. As a negative control we stained the cells either with primary or secondary antibody alone.

2.8. Data Analysis

Data are expressed as mean ± SEM. Comparisons between groups were made either by Student's t-test or one way ANOVA followed by Tukey's post-hoc test where appropriate (GraphPad Software, Inc. San Diego, CA), and groups were considered significantly different if p< 0.05.

3. RESULTS

We examined the effect of H2O2 (a direct oxidant, 100 μm) and BSO (a pro-oxidant drug, 1mM) treatment on oxidative stress markers including protein carbonylation and malondialdehyde (MDA) in CATH.a cells. Both H2O2 and BSO significantly increased protein carbonylation (Figure 1A) and MDA (Figure 1B) with increasing time as compared to vehicle treated cells. H2O2 caused 123% increase in protein carbonylation and 240% increase in MDA at 0.5h and 207% increase in carbonylation and 280% increase in MDA at 1h. BSO resulted in 174% increase in protein carbonylation and 260% increase in MDA at 0.5h and 300% increase in carbonyls and 730% increase in MDA at 1h. Minimum cell death or morphological deterioration was noted at the dose of H2O2 or BSO used (Figure1 C,D). Cell viability was detected at 91–94% with either treatment. Next, mRNA expression of several RGS proteins in CATH.a cells were examined using semi-quantitative RT-PCR with specific primers for each RGS protein (Figure. 2A). It appears that only RGS2 mRNA levels increased upon H2O2 (100 μm for 1h) or BSO (1mM for 1h) treatment (Figure. 2B). This was confirmed using quantitative-RTPCR which also indicate that RGS2 mRNA levels are significantly upregulated in response to induction of oxidative stress via both, H2O2 (+200%) or BSO (+300%) treatment as compared to vehicle treated controls (Figure. 3A). The related RGS protein, RGS4 mRNA expression remained unchanged in response to both H2O2 and BSO (Figure. 3B). We also examined the effect of H2O2 and BSO treatment on mRNA expression of GLO1 and GSR1. A significant increase in GLO1 (+300%: H2O2; +310%: BSO) and GSR1 (+333%: H2O2; +280%: BSO) mRNA expression was observed (Figure. 3A).

Fig. 1. Measurement of oxidative stress and examination of cell viability and morphology.

Fig. 1

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CATH.a cells were treated with vehicle or 100 μM H2O2 or 1mM BSO for 30 and 60 min. MDA was quantified using molar extinction coefficient 1.56×105 M/cm [1,2]. Protein carbonylation was measured using an ELISA based assay (Cell Biolabs Inc. USA). *significantly different from respective vehicle, p<0.05, N=4. Cell viability was measured using trypan blue exclusion method. CATH.a cells were treated with vehicle/H2O2/BSO as before, harvested by trypsinization, stained with trypan blue and counted using a haemocytometer. Cell viability was expressed as % of excluded divided by total number of cells. N=3. Cell morphology was examined using Olympus 1×81 Fluorescence Deconvolution Microscope System under bright field (phase contrast setting). Shown is a representative area of cells at the indicated treatment.

Fig.2. Effect of H2O2 or BSO treatment on mRNA expression of RGS proteins in CATH.a cells.

Fig.2

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Total RNA was isolated from CATH.a cells, reverse transcribed and amplified with primers for specific RGS proteins [19] using semiquantitative-RTPCR [17] (A). CATH.a cells were treated with vehicle (V), H2O2, (100 μM:H) or BSO (1 mM:B) for 1h, RNA was isolated and reverse transcribed using RTPCR (B). PCR products were electrophoresed on a 2% agarose gel, stained with 0.5μg/ml ethidium ethidium bromid bromide, and visualized under UV. A molecular ruler (100–3000 bp) on the left-hand side of the gel is used for reference.

Fig.3. H2O2 or BSO treatment increases mRNA expression of RGS2, GLO1 and GSR1 in CATH.a cells.

Fig.3

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CATH.a cells were treated with H2O2 (100 μM) or BSO (1mM) for 1h and mRNA levels were determined by real-time PCR using TaqManR one-step RT-PCR kit using primer sets specific for RGS2, RGS4, GLO1, GSR1 and GAPDH primers. Total mRNA used in PCR reaction was 50 ng. GAPDH was used as an internal control. *Significantly different from respective vehicle, p<0.05, N=6.

Furthermore, CATH.a cells were transfected with RGS2 antisense or random oligonucleotides (2.5mM) and cells treated with vehicle/H2O2 (100 μM) for 1h. RGS2 antisense treatment resulted in a significant decrease in H2O2-induced GLO1 (−181%) and GSR1 (−300%) mRNA expression as compared to random oligonucleotide treated vehicle or H2O2 samples (Figure 4A). Random RGS2 oligo treatment had no effect on H2O2-induced RGS2, GLO1 and GSR1 mRNA expression suggesting specificity for RGS2 antisense oligos. Moreover, H2O2-induced GLO1 and GSR1 protein expression also was significantly reduced (−140%, −200% respectively) when compared to random oligonucleotide transfected vehicle controls (Figure 4B,C). Protein expression of two other antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) remained unchanged with H2O2 or with RGS2 antisense treatment (Figure 4D,E).

Fig.4. Pretreatment of cells with RGS2 antisense attenuated H2O2-induced increase in GLO1 and GSR1.

Fig.4

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CATH.a cells were treated with RGS2 random or antisense oligonucleotides (2.5 mM) using FugGENE6 reagent [16]. 48h later, cells were treated with H2O2 (100 μM;1h) and harvested, RNA isolated and processed for q-RTPCR (A). Similarly treated cells were processed for western blotting and immunoblotted for GLO1, GSR1, SOD and CAT (B–E). Protein densities were normalized to respective GAPDH density (internal control). *Significantly different from both random oligo and RGS2 AS transfected vehicle treated cells, #from H2O2-treated cells, p<0.05, ANOVA, N=3.

Induction of oxidative stress caused activation of PKC determined in CATH.a cell lysates treated with H2O2 (100 μM). PKC activity was significantly increased by ~50% after 15 min of H2O2 treatment as compared to vehicle treated samples. The activation however was noted maximum at 1h of H2O2 treatment (+210%) (Figure. 5A). Nuclear translocation of transcription factor, Sp-1 also was determined in response to H2O2 (100 μM, 1h) treatment. We observed that H2O2 treatment resulted in translocation of Sp-1 from the cytosol to the nucleus (Figure. 5B). In vehicle-treated cells, Sp-1 (red signal) was dispersed throughout the cytosol with almost no nuclear localization (blue signal) (Figure. 5B). In cells treated with H2O2, the intensity of the Sp-1 signal in the nuclei increased dramatically, staining the entire nucleus red. Moreover, as nuclear localization of Sp-1 increased, the Sp-1 signal disappeared from the cytosol. Similar results were obtained for BSO (data is shown for H2O2 only). We also detected increased Sp-1 protein accumulation in nuclear extracts of CATH.a cells treated with H2O2 as compared to vehicle-treated cells. Nuclear accumulation of Sp-1 proteins was increased by ~80% (expressed as % of control) (Figure. 5C). PKC activation also resulted in nuclear translocation (Figure. 5D) and nuclear accumulation of RGS2 (Figure. 5E). A considerable amount of RGS2 protein was detected at the basal level in the nuclei.

Fig.5. H2O2 treatment activates PKC and increases nuclear translocation of Sp-1 and RGS2.

Fig.5

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PKC activity was measured using a kit based assay [18] after H2O2 treatment (100 μM;0.25–4h) in CATH.a cells (A). Immunofluorescence staining for Sp-1 and RGS2 after 1h of vehicle (upper panel) and H2O2 (lower panel) treatment was determined [17] using Sp-1 (B) and RGS2 (D) primary and Cy3 conjugated secondary antibodies. Shown are representative images and overlays of at least 5–6 fields on the same slide of Sp-1/RGS2 (visualized by cy3 filter, red), DAPI (staining for cell nuclei visualized with DAPI filter, blue) and overlay of the two images (right plates, B, D) under an oil immersion objective (×60, 1.4NA, scale bar:10 μm)) on an Olympus 1×81 Fluorescence Deconvolution Microscope System. Nuclear abundance of Sp-1 (C) and RGS2 (E) was detected by western blotting in extracted nuclear fractions of CATH.a cells, normalized to lamin B (loading control) and presented as %control [17]. Upper panel: representative blots (V, vehicle; H, H2O2). Lower panel: densitometric analysis. *Significantly different from vehicle, p<0.05, t-test, N=3.

Furthermore, H2O2 treatment (100 μM) significantly activated p38 MAPK pathway at 15 min detected via western blotting wherein H2O2 treatment significantly increased (+100%) phospho-p38 protein expression (normalized to total protein) in CATH.a cells. This activation was sustained until 4h of H2O2 treatment (Figure. 6A). p38 MAPK activity is measured as an index of its phosphorylation. H2O2-induced increase in p38 MAPK phosphorylation was prevented when cells were pretreated with p38 MAPK inhibitor, SB203580 (cat# 559389, EMD4Biosciences, USA, 25 μM, 1h). SB203580 was added 1h prior to H2O2 treatment and also was present during H2O2 incubation (Figure. 6B). Thus, our treatment conditions sufficiently block H2O2-induced p38 activation. SB203580 at the concentration used is reported to affect RGS2 transcripts in C6 astrocytoma cells [13] and at this concentration, other MAPK proteins (phospho and total ERK1/2) were not altered (data not shown). Furthermore, activation of p38 significantly increased mRNA expression of RGS2 (+340%) and pretreatment of cells with the p38 inhibitor prevented the increase in RGS2 expression. RGS4 mRNA expression remained unchanged (Figure. 7A). Interestingly, H2O2-induced increase in GLO1 and GSR1 mRNA expression also is prevented with SB203580 or with PKC inhibitor, chelethyrine chloride (CC) (10 μM, 2h) treatment (Figure. 7B). The level of GLO1 and GSR1 mRNA expression is reduced further when cells were treated with SB203580 and CC in combination.

Fig.6. Oxidative stress activates p38 MAPK.

Fig.6

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CATH.a cells were treated with or without p38 inhibitor 1h prior to vehicle/H2O2 treatment (0.25–4h). Cells were harvested and subjected to western blotting using anti-P-38 and p38 antibodies. Bands of P-38 were normalized with total p38 protein and values expressed as densitometric ratios between the two. Lanes 1: vehicle; 2: SB203580 (25 μM;), 3: H2O2 (100 μM/1h); 4: SB203580+H2O2. SB20358 was present before (1h) and during H2O2 incubation (A). Lanes 1:vehicle; 2–4: H2O2 (100 μM for 0.25, 1, 4h respectively) (B). *Significantly different from vehicle (A) or SB203580 vehicle treated (B) cells, #from H2O2-treated cells. p<0.05, ANOVA, N=3.

Fig.7. Differential effect of PKC and p38 inhibitors on mRNA expression of RGS2, GLO1 and GSR1.

Fig.7

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CATH.a cells were treated with or without p38 inhibitor (25 μM, 1h) or chelethyrine chloride (CC) (10 μM, 2h) or both prior to vehicle/H2O2 (100 μM, 1h) or BSO (1 mM, 1h) treatment. Cells were harvested and processed for q-RTPCR and levels of RGS2, RGS4 (A), GLO1 and GSR1 (B) mRNA determined. *Significantly different from vehicle, #from H2O2 or BSO, p<0.05, ANOVA, N=4.

Finally, the wild type RGS2 promoter [20] exhibited increased luciferase activity while the mutant RGS2 promoter [20] in which Sp-1 site is mutated, remained unresponsive to H2O2 or BSO treatment and failed to increase luciferase activity. Pretreatment with p38 inhibitor, SB203580 inhibited BSO or H2O2 induced RGS2 promoter activity (Figure. 8). Similar results were obtained in BE(2)-C cells, a human neuroblastoma cell line.

Fig.8. Oxidative stress increases RGS2 promoter activity in a p38 dependent manner.

Fig.8

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CATH.a cells were transfected with WT or MUT RGS2 luciferase reporter constructs. 48h later, cells were treated with H2O2 (100 μM/1h)- or BSO (1 mM/1h) and luciferase (LUC) activity was measured. Results are mean±SEM of ratios of luminescence of firefly and renilla (transfection control) luciferase and normalized to protein. *Significantly different from vehicle, #from H2O2 or BSO, p<0.05, ANOVA, N=4.

4. DISCUSSION

In this study, RGS2 mRNA levels were selectively and significantly upregulated in response to H2O2 or BSO-induced oxidative stress in CATH.a cells. Interestingly, both mRNA and protein levels of GLO1 and GSR1 also increased with induction of oxidative stress. Furthermore, pretreatment of CATH.a cells with RGS2 antisense, attenuated H2O2-induced increase in GLO1 and GSR1 levels. Expression of two other antioxidant enzymes, CAT and SOD remained unchanged. It seems reasonable to suggest that upregulation of GLO1 and GSR1 expression in response to oxidative stress is quite possibly an RGS2-dependent event as reducing RGS2 expression by antisense treatment prevented the increase in GLO1 and GSR1 mRNA and protein expression. Whether GLO1 and GSR1 can be considered as potential transcriptional targets of RGS2 remains to be validated in future.

Our study suggests that increase in RGS2 expression is p38-dependent while transcriptional activation of GLO1 and GSR1 appears to require simultaneous activation of both p38 and PKC pathways. This becomes clear from the observation that H2O2 or BSO-induced oxidative stress increased mRNA expression of RGS2 while pretreatment with p38 inhibitor prevents this increase. Increase in GLO1 and GSR1 mRNA upon induction of oxidative stress is significantly reduced with p38 inhibitor treatment. Pretreatment of CATH.a cells with PKC inhibitor, chelethyrine chloride (CC) also prevents H2O2-induced increase in GLO1 and GSR1 but RGS2 mRNA expression remains unchanged. In the presence of both inhibitors, SB203580 and CC, H2O2-induced increase in GLO1 and GSR1 is most potently reduced when compared to inhibitor treatment done separately.

Involvement of p38 pathway in increasing RGS2 mRNA expression in an Sp-1 dependent manner has been reported [20]. Relevant to this we observed that, H2O2 activated p38 MAPK pathway and increased Sp-1 nuclear accumulation and translocation. The p38 pathway has been reported to phosphorylate Sp-1 [21] and an Sp-1 binding site is present in mouse RGS2 promoter [20] suggesting that Sp-1 binding to the RGS2 promoter increases its expression. Our data supports this notion. Moreover, oxidative stress activated PKC pathway. PKC is reported to phosphorylate RGS2 [22] and RGS2 is known to translocate to nucleus [23]. These reports are in agreement with our finding of nuclear translocation of RGS2 with induction of oxidative stress. We postulate that RGS2 upon PKC phosphorylation is shuttled to the nucleus, where it interacts with transcription factor NF-E2-related factor (Nrf)-2, and this complex then binds to Anti-oxidant Response Elements (ARE) present in GLO1 and GSR1, to increase transcription. Our postulation is made more compelling by the fact that PKC phosphorylation of Nrf2 is reported to release Nrf2 from the inhibitory protein (INrf2). Nrf2 then translocates to the nucleus, forms heterodimers with unknown proteins, and activates transcription of genes containing ARE [24–26]. Whether RGS2 acts as a cofactor, an activator or a binding partner for Nrf2 remains to be investigated. Therefore, it is reasonable to conclude that upon induction of oxidative stress, both PKC and p38 pathways are activated. p38 phosphorylates and activates Sp-1. Sp-1 binds to the RGS2 promoter and activates its expression. PKC induces nuclear translocation of RGS2 and facilitates interaction with Nrf2, which induces ARE binding and promotes transcriptional activation of GLO1 and GSR1.

5. ACKNOWLEDGEMENTS

Funding: GEAR grant, UH (S.S), NIH/NIA AG29904 (M.A), Academy of Finland NEURO research program and Academy Research Fellowship (I.H), Taiwan National Science Council grant NSC 97-2320-B-010-018-MY2 (Y-L.W).

Abbreviations

(RGS)-2

Regulator of G-protein Signaling Protein

(LC)

locus coeruleus

Footnotes

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