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Keywords:

  • antioxidant responsive element;
  • NQO1;
  • oxidative stress;
  • primary cortical neuronal cultures;
  • tBHQ;
  • transgenic reporter mice

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Many phase II protective genes contain a cis-acting enhancer region known as the antioxidant response element (ARE). Increased expression of these genes contributes to the protection of cells from oxidative stress. Transgenic reporter mice were created that carry in their genome the core ARE coupled to the human placental alkaline phosphatase (hPAP) reporter gene. Primary cortical cultures derived from these mice were treated with tBHQ resulting in a dose-dependent increase in hPAP activity. Histochemical staining for hPAP activity was observed in both glia and neurons from tBHQ-treated cultures. The tBHQ-mediated increase in hPAP was not affected by the antioxidant glutathione monoethyl ester (GSHEE), whereas the increase in hPAP following DEM treatment was completely blocked by GSHEE. Pre-treatment of cultures with the PI3-kinase inhibitor LY 294002 demonstrated a dose-dependent decrease in tBHQ-induced hPAP activity. In addition, the tBHQ-mediated expression of ARE-driven genes in primary cortical cultures was blocked by LY 294002. Interestingly, basal expression of Nrf2 was also inhibited by LY 294002. We theorize that increased levels of genes controlled by the ARE are important for cellular protection against oxidative stress. These ARE-hPAP transgenic mice will be an important in vivo model for testing our hypothesis.

Abbreviations used:
AraC

cytosine arabinoside

ARE

antioxidant response element

BHA

butylated hydroxyanisole

DEM

diethyl maleate

dtBHQ

di-tert-butylhydroquinone

GCLR

gamma-glutamylcysteine ligase regulatory subunit

GCLC

gamma-glutamylcysteine ligase catalytic subunit

GSH

glutathione

GSHEE

glutathione monoethyl ester

HO-1

heme oxygenase 1

hPAP

human placental alkaline phosphatase

NQO1

NAD(p)H:quinone oxidoreductase 1

PI3-kinase

phosphatidylinositol-3-kinase

ROS

reactive oxygen species

tBHQ

tert-butylhydroquinone.

A cell's ability to protect itself from oxidative stress caused by the toxic accumulation of reactive oxygen species (ROS) and electrophiles, relies, in a large part, on the levels of phase II protective enzymes. The 5′ flanking region of many of these protective genes contain a cis-acting enhancer region known as the antioxidant response element (ARE). These include, but are not limited to, heme oxygenase-1 (HO-1), glutathione-S-transferases (GSTs), gamma-glutamylcysteine ligase (catalytic and regulatory subunits; GCL) and NAD(P)H:quinone oxidoreductase 1 (NQO1; Li and Jaiswal 1992; Mulcahy and Gipp 1995; Prestera et al. 1995; Moinova and Mulcahy 1998). The ARE was first identified in 1990 by Rushmore and Pickett (1990) as a regulatory element in the upstream region of the rat GST-Ya subunit. The core sequence is 5′-GTGACnnnGC-3′. It was later demonstrated that both the basal and inducible activity of NQO1 by planar aromatics, phenolic antioxidants and H2O2, was mediated through the ARE (reviewed in Dinkova-Kostova and Talalay 2000). Furthermore, it has been demonstrated that Nrf2 regulates the gene expression of phase II detoxifying enzymes through binding to the ARE (Venugopal and Jaiswal 1996; Itoh et al. 1999; Moinova and Mulcahy 1999; Wild et al. 1999).

The experiments defining ARE activation are based primarily on studies in hepatoma cell lines. NQO1, however, is located in most tissues, including brain as was seen by immunohistochemical-positive staining in rat glial cells in the forebrain (Schultzberg et al. 1988), Bergmann radial glial cells in the cerebellum (Schultzberg et al. 1988), and glial cells in human brains (Wang et al. 2000). Murphy et al. (1991) showed that pre-treatment of a neuroblastoma cell line with tert-butylhydroquinone (tBHQ), a known activator of the ARE, significantly attenuated glutamate toxicity by 80%. More recently it was demonstrated that pre-treatment of the N18-RE-105 neuroblastoma-retina hybridoma cell line with a different ARE activator, dimethyl fumarate (DMF), protected against dopamine and H2O2 toxicity. This protective effect was attributed to elevated intracellular GSH and increased activities of NQO1 and GST. A stable cell line overexpressing NQO1 and GST, however, was not resistance to dopamine toxicity (Duffy et al. 1998). This suggests that these two enzymes alone were not sufficient in protecting against dopamine toxicity, but that the coordinate induction of genes regulated by the ARE may have provided the mechanisms necessary for cell protection and survival.

In primary cultures of cerebellar glial cells and in a human neuroblastoma cell line, our laboratory has defined the core ARE sequence containing a complete 5′ palindrome and conserved 3′ GC nucleotide pair that is required for maximal activation by tBHQ (Ahlgren-Beckendorf et al. 1999; Moehlenkamp and Johnson 1999). Activation of the ARE in both culture systems was correlated with increased NQO1 activity. We have also shown that activation of the ARE in IMR-32 human neuroblastoma cells treated with tBHQ and diethyl maleate (DEM) was dependent upon the transcription factor, Nrf-2 (Lee et al. 2001a). Interestingly, activation of the ARE by tBHQ was independent of oxidative stress (Lee et al. 2001a). In addition, it was demonstrated that activation of the ARE in these human neuroblastoma cells was through a phosphatidylinositol-3-kinase (PI3-kinase) dependent pathway that lies upstream of Nrf-2 (Lee et al. 2001b).

To further understand how the ARE is controlled in primary cultures and in vivo, we have created two founder lines of transgenic reporter mice. Fifty-one basepairs of the rat NQO1 promoter region containing the ARE coupled to the human placental alkaline phosphatase (hPAP) reporter gene have been inserted into the genome of these mice. The studies presented in this manuscript were aimed at determining the cell-specific patterns of ARE activation in primary cortical cultures derived from the ARE-hPAP transgenic mice and if tBHQ-mediated activation of the ARE was associated with oxidative stress or PI3-kinase.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Generation of ARE reporter mice

A 51 basepair segment of rat NQO1 promoter containing the core ARE was inserted into a TATA-Inr minimal promoter : heat-stable human placental alkaline phosphatase (hPAP) reporter gene construct. Linearized construct was microinjected into fertilized oocytes that were then transferred to pseudopregnant mothers. The resulting pups were tail-clipped and ear-punched at 21 days of age for genotyping and identification, respectively. DNA was extracted from tail clips using the Qiagen DNeasy Kit (Qiagen, Valencia, CA, USA) according to the manufacture's protocol. PCR was used to identify positive animals. For the NQO1-ARE-hPAP transgene, the sense primer is 5′-CTAGAGTCACAGTGACTTGGCAAA-3′ and includes the ARE core (bold letters). The antisense primer, 5′-GGAAGATGATGAGGTTCTTGGCGG-3′, is located 185 basepairs (bp) downstream from the translation start site of hPAP. The PCR product is 458 bp in length. From these original founder mice, we have successfully established two lines of ARE transgenic reporter mice.

Primary cortical cultures

Embryos were removed on E15–E16 and transferred to ice-cold Mg2+- and Ca2+-free Hank's balanced salt solution (HBSS, Gibco, Life Technologies, Rockville, MD, USA). Cortical tissue was carefully removed, placed in 5–10 mL ice-cold Mg2+- and Ca2+-free HBSS, gently triturated to break tissue into medium/small pieces and centrifuged at 300 g for 2 min. HBSS was removed and 10 mL of 0.05% (wt/vol) trypsin was added in HBSS (200 µL of a 10 × stock in 10 mL of HBSS). Tissue was digested in a shaking water bath at 37°C for 10 min and then centrifuged at 300 g for 2 min. Cells were washed twice with 10 mL of HBSS and 4 mL of CEMEM [390 mL of EMEM (Mediatech, Inc., Herdon, VA, USA), 50 mL of heat-inactivated FBS (Atlanta Biologicals, Norcress, GA, USA), 50 mL of heat-inactivated horse serum (Atlanta Biologicals), 5 mL L-Gln (FAC = 4.0 mm), 5 mL of Pen (FAC = 100 IU/mL)/Strep (FAC = 100 µg/mL)] was added to the cell pellet. Cells were gently triturated 10–20 times with a 10-mL pipette to generate a single cell suspension that was filtered through a 70-µm mesh filter. Cell viability was determined by trypan blue exclusion. Cells were plated in poly d-lysine (Sigma Chemical Co., St Louis, MO, USA)-coated dishes and allowed to attach for 30 min at 37°C (5% CO2) after which the medium was replaced. Cells were grown and maintained in a humidified environment at 5% CO2 and 37°C. Forty-eight hours after plating, medium was changed and, in some cases, cells were treated with cytosine arabinoside (AraC, FAC = 4 µm, Sigma Chemical Co.) to prevent non-neuronal cell proliferation.

In experiments where hPAP activity was measured, cells were grown in 96-well plates and seeded at a density of 100 000 cells in 100 µL. After 3 days in vitro, fully differentiated cultures were treated for 72 h with vehicle (0.03% (v/v) ethanol), tBHQ (Acros Organics, Pittsburg, PA, USA), butylated hydroxyanisole (BHA, Acros Organics), di-tert-butylhydroquinone (dtBHQ, Acros Organics), or DEM (Aldrich, Milwaukee, WI, USA). To determine the effect of the presence of an antioxidant, the cultures were pre-treated with vehicle (PBS) or 2.0 mm glutathione monoethyl ester (GSHEE) for 2 h prior to treatments with tBHQ, BHA, dtBHQ, or DEM. For experiments using a PI-3 kinase inhibitor, cultures were pre-treated with vehicle (0.3% (v/v) ethanol) or LY 294002 (Calbiochem, San Diego, CA, USA) for 20 min prior to treatment with vehicle (0.03% (v/v) ethanol) or tBHQ.

For the histochemical analysis of hPAP or NQO1 activity, cells were plated in 8-well chamber slides at a density of 240 000 cells in 400 µL in each well and treated on day 3 with tBHQ for 72 h. Lastly, for the RT-PCR experiments, cells were cultures in 60-mm dishes, 8 000 000 cells in 8 mL were plated in each dish and pre-treated with vehicle (0.3% (v/v) ethanol) or LY 294002 for 20 min prior to treatment with vehicle (0.03% (v/v) ethanol) or tBHQ for 24 h.

NQO1 assay and histochemistry

Whole-cell extracts were prepared by lysing cells in the 96-well plate. NQO1 activity was measured by monitoring the reduction of MTT (610 nm) coupled to menadione in the presence or absence of dicumarol (Lind et al. 1990). Data are presented as the change in absorbance per minute. Unpaired Student's t-tests were used to compare statistical significance between treatment groups. To visualize NQO1 activity, cells were fixed in 3% (wt/vol) paraformaldehyde for 20 min, washed with PBS and pre-incubated with reaction buffer [25 mm Tris, pH 7.4, 0.08% (v/v) Triton X-100, 2 mg/mL (bovine serum albumin, BSA) for 30 min in the presence or absence of 100 µm dicumarol (NQO1 inhibitor)]. The pre-incubation solution was replaced with reaction buffer ± dicumarol containing 100 µm NBT (Calbiochem) and 100 µm LY 83583 (NQO1 substrate, Calbiochem). The staining reaction was initiated by adding NADPH (1.0 mm final concentration). The reaction was incubated at 37°C for 30 min. Specificity was determined by lack of staining in the presence of dicumarol or absence of LY 83583 (Murphy et al. 1998).

HPAP assay and histochemistry

Whole-cell extracts were prepared by lysing cells in 96-well plates. HPAP levels were quantified by measuring alkaline phosphatase activity. Briefly, cells were lysed in a Tris-5 mm magnesium saline solution containing 1% (wt/vol) CHAPS (TMNC buffer, Sigma). Extracts were incubated at 65°C for 30 min to heat inactivate endogenous alkaline phosphatase activity, then incubated at room temperature in the presence of a chemiluminescent substrate for alkaline phosphatase (Phospho-light; Tropix, Bedford, MA, USA). The resulting luminescent signal represents relative hPAP activity. Unpaired Student's t-tests were used to compare statistical significance between treatment groups. To visualize hPAP activity, fixed cells [3% (wt/vol) paraformaldehyde for 20 min were incubated in a 50 mm Tris−5 mm magnesium saline solution (TMN), pH 10, at 65°C for 15 min], to heat inactivate any endogenous alkaline phosphates activity. Cells were then stained for hPAP by replacing the TMN with a staining solution containing 1 mg/mL of NBT and 1 mg/mL X-phosphate (5-bromo-4-chloro-3-indoyl-phosphate; Calbiochem) and incubating at 37°C for 30–45 min.

Glutathione levels

Total glutathione levels (GSH and GSSG) were determined using a modified Tietze (1969) method by spectrophotometrically measuring the reduction of GSH (Sigma) by dithionitrobenzoic acid (DTNB, Sigma) coupled to the recycling of GSSG to GSH with glutathione reductase. Primary cortical cultures were plated as described above in 6-well dishes at a density of 2.5 million cells per well and were pre-treated for 2 h with 2.0 mm GSHEE or vehicle (water). Cultures were then treated with vehicle [0.015% (v/v)], dimethylsulfoxide (DMSO), 30 µm tBHQ, or 30 µm DEM for 4 h after which the cells were washed with PBS, lysed with cold 3% perchloric acid, and centrifuged at 4°C at 7400 g. The supernatants were diluted with 9 volumes of 0.1 m Na2HPO4. The neutralized supernatant was aliquoted into 96-well plates containing 0.1 m potassium phosphate buffer (pH 7.5) with 0.1 mg/mL DTNB and 0.32 mg/mL NADPH. Using a microtiter plate reader, total glutathione was measured as the change in absorbance at 405 nm per minute upon the addition of GSSG reductase (Calbiochem) at 8 units/mL. Total glutathione concentrations were determined from standard curves.

RT-PCR

Total RNA was isolated from primary neuronal cultures by Trizol Reagent (Life Technologies, Gibco) following manufacturer's instructions. One microgram of RNA was reverse transcribed using primer for the hPAP gene or Oligo (dT)15 primer in accordance with the RT System by Promega (Madison, WI, USA). The resulting cDNA was then amplified by PCR using primer sets for the genes: hPAP (forward 5′-TTGCCCCAAATCTCAACTTC-3′; reverse 5′-TGGGTAGCTGGGACTACAGG-3′) resulting in a PCR product of 351 basepairs; NQO1 (forward 5′-CATTCTGAAAGGCTGGTTTGA-3′; reverse 5′-TTTCTTCCATCCTTCCAGGAT-3′) resulting in a PCR product of 300 basepairs; GCLR (forward 5′-ACCTGGCCTCCTGCTGTGTG-3′; reverse 5′-GGTCGGTGAGCTGTGGGTGT-3′) resulting in a PCR product of 290 basepairs; Nrf2 (forward 5′-TCTCCTCGCTGGAAAAAGAA-3′; reverse 5′- AATGTGCTGGCTGTGCTTTA-3′) resulting in a PCR product of 430 basepairs; and β-Actin (forward 5′-CCCAGAGCAAGAGAGGTATC-3′; reverse 5′-AGAGCATAGCCCTCGTAGAT-3′) resulting in a PCR product of 340 basepairs.

Results

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Expression of hPAP and QR in primary neuronalcultures treated with tBHQ

Primary cortical cultures derived from the ARE-hPAP transgenic mice were treated with increasing doses of tBHQ to determine if the cells were capable of expressing the hPAP reporter gene. As shown in Fig. 1(a), hPAP activity in the cell lysate from these cultures demonstrated a dose-dependent increase by tBHQ resulting in 22–25-fold activation at 50 µm and 100 µm. These results confirm that the mice are capable of expressing the hPAP reporter gene. The increase in hPAP activity was attributed to the increase in the activation of the ARE by tBHQ and was supported by coordinate induction of NQO1 (Fig. 1b) which also increased in a dose-dependent fashion. Similarly, hPAP and NQO1 activities in cultures not treated with AraC were induced 13-fold (vehicle, 95 497 ± 4341; tBHQ, 1243 415 ± 51 448) and fourfold (vehicle, 0.0029 ± 0.0002; tBHQ, 0.0128 ± 0.0011), respectively.

image

Figure 1. TBHQ dose response in primary cortical cultures derived from ARE-hPAP transgenic mice. A hemizygous ARE-hPAP mouse was crossed with a control mouse (B6/SJL). Primary cortical tissue from E16 embryos was harvested and cultured in 96-well poly d-lysine-coated plates as described in Experimental Procedures. Forty-eight hours after plating, cultures in the presence of cytosine arabinoside were treated with tBHQ for 72 h. (a) Cultures were treated with increasing doses of tBHQ as indicated. Whole-cell extracts were then analyzed for hPAP activity as described above. Values represent the mean ± SEM (n = 5), resulting in 20–24-fold inductions by tBHQ at its highest concentration compared to vehicle treated cultures. (b) Cultures were treated with increasing doses of tBHQ as indicated. NQO1 activity was measured in whole cell extracts as described in the Experimental Procedures. Data are presented as the mean change in absorbance per min ± SEM (n = 5). Fold activations by tBHQ- versus vehicle-treated cultures were sixfold. (c) Primary cortical cultures were derived from founder line #1 (FF1), founder line #15 (FF15) or a control animal (non-transgenic) and treated with vehicle (0.03% (v/v) ethanol) or 50 µm tBHQ. Whole cell extracts were analyzed for hPAP activity as described in Experimental Procedures. Each value represents the mean ± SEM. Note that values for hPAP activity in cultures from control animals were very low and not distinguishable from background.

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Our laboratory has created two founder lines of ARE-hPAP transgenic mice that we have labeled FF1 and FF15. Primary cortical cultures from both founder lines and the non-transgenic control line (B6/SJL) were treated with vehicle (0.03% (v/v) ethanol) or 50 µm tBHQ. Lysates from control cultures showed no hPAP activity above background in either vehicle- or tBHQ-treated cultures. Lysates from cortical cultures derived from FF1 and FF15 founder lines showed low basal activity that was induced by 50 µm tBHQ to 21- and 28-fold, respectively (Fig. 1c). Differences in relative hPAP activity between the two lines were attributed to the cultures being derived from embryos whose parents were heterozygous for the hPAP reporter gene. Hence, in these sets of experiments, lysates from founder line FF15 that resulted in higher levels of hPAP activity had more hPAP-positive embryos than FF1. This was verified by histochemical staining for hPAP of the individual cultures and PCR of genomic DNA from the tails of each litter (data not shown). The expression and induction of the hPAP, however, was similar and the remaining experiments were conducted using either founder line.

Histochemical analysis for hPAP and NQO1 activityin primary cortical cultures

To determine cell-specific activation of the ARE, primary cortical cultures derived from the ARE-hPAP reporter mice were stained for hPAP activity (Fig. 2a–d). Cultures were grown in the presence and absence of AraC to control for non-neuronal cell proliferation and subsequently treated with vehicle [0.03% (v/v) EtOH] or 50 µm tBHQ for 72 h. In cultures grown without AraC, the resulting cell population was a mixed culture system consisting of glia and neurons. In cultures grown in the presence of AraC, neurons were the most abundant cell type. Immunohistochemical staining of these culture systems with antibodies to glial fibrillary acidic protein (GFAP) identified cells as astrocytes, while antibodies to microtuble-associated protein 2 (MAP-2) identified cells as neurons (data not shown). Basal expression of hPAP was isolated to astrocytes in both the presence and absence of AraC. Vehicle treatment had no affect on the basal pattern of expression. TBHQ treatment for 72 h resulted in a dramatic increase in hPAP activity in astrocytes (Fig. 2b, arrow) as well as neurons (Fig. 2d, arrow). The staining intensity of neurons varied and the most intense neuronal staining was observed in neurons lying in close proximity to astrocytes. This suggests that the astrocytes and/or glia may contribute to the extent of activation of the ARE in neurons (Fig. 2c,d). No staining was detected in heat-inactivated cultures derived from transgene-negative embryos (data not shown).

image

Figure 2. HPAP histochemistry in primary cortical cultures treated with 50 µm tBHQ. Images shown here are representative of cultures that were harvested and treated with vehicle (0.03% (v/v) ethanol) or 50 µm tBHQ for 72 h. Cultures were stained for hPAP activity as described Experimental Procedures. Panels (c) and (d) have been treated with AraC. The arrow in panel (a) indicates low basal activity in astrocytes from vehicle treated cultures, while the arrow in panel (b) indicates intensified staining in astrocytes from tBHQ-treated cultures. Similarly, the arrow in panel (c) indicates low or no basal activity in neurons (background) from vehicle-treated cultures, while the arrow in panel (d) indicates significant neuronal staining in tBHQ-treated cultures. Scale bar: 40 µm.

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Similar results were seen for NQO1 staining in cultures treated as described above (Fig. 3a–d). Low basal activity and significant induction in astrocytes and neurons were noted in the same ARE-hPAP-positive cells. NQO1 staining was blocked by the addition of dicoumarol or removal of the NQO1 substrate, LY 83583 (data not shown).

image

Figure 3. NQO1 histochemistry in primary cortical cultures treated with 50 µm tBHQ. Images shown here are representative of cultures that were harvested and treated with vehicle [0.03% (v/v) ethanol] or 50 µm tBHQ for 72 h. Cultures were stained for NQO1 activity as described Experimental Procedures. Panels (c) and (d) have been treated with AraC. The arrow in panel (a) indicates low basal activity in astrocytes from vehicle-treated cultures, while the arrow in panel (b) indicates intensified staining in astrocytes from tBHQ-treated cultures. Similarly, the arrow in panel (c) indicates low or no basal activity (background staining) in neurons from vehicle treated cultures, while the arrow in panel (d) indicates significant neuronal staining in tBHQ-treated cultures. Scale bar: 40 µm.

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Effect of GSHEE on the induction of hPAP in primary neuronal cultures from ARE-hPAP transgenic mice

We have previously demonstrated that activation of the ARE by tBHQ in human neuroblastoma cells was independent of oxidative stress. Pre-treatment with antioxidants or antioxidant enzymes did not block the tBHQ-mediated ARE activation yet blocked the DEM-mediated ARE activation (Lee et al. 2001a). We designed similar experiments to examine the effects of pre-treating primary cortical cultures derived from the ARE-hPAP reporter mice with the antioxidant GSHEE, a cell permeable form of glutathione. In addition, we analyzed various compounds to determine what structures or moieties may be important in the regulation of the ARE. HPAP activities in the presence of GSHEE were consistently lower than in the absence of GSHEE and in most cases this was significant. Results indicated that pre-treatment of primary cortical cultures with 2 mm GSHEE had no significant impact on the tBHQ (30 µm) activation of the ARE as measured by hPAP activity (Fig. 4a). However, GSHEE significantly inhibited DEM activation of the ARE in the same culture system (Fig. 4a). Other compounds we tested in our system included BHA, a known food antioxidant and the parent compound to tBHQ, and dtBHQ, a derivative of tBHQ that has similar oxidative properties (Kahl et al. 1989; Okubo et al. 1997). Neither BHA nor dtBHQ at 30 µm activated the ARE and values indicate hPAP activity similar to that of basal activity (data not shown).

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Figure 4. Effects of GSHEE on ARE activation in primary cortical cultures treated with tBHQ or DEM. (a) Primary cortical cultures were plated in a 96-well plate format and pre-treated with 2 mm GSHEE or vehicle for 2 h followed by treatments with 30 µm tBHQ or increasing doses of DEM for 72 h. Relative hPAP activity was measured in the cell lysates as described above. The data are expressed as mean relative luminescent units ± SEM (n = 3). (b) Primary cortical cultures were plated in 6-well plate format and pre-treated with 2 mm GSHEE for 2 h followed by treatments with 30 µm tBHQ or 30 µm DEM for 4 h. Total glutathione (GSH and GSSG) levels were measured in cell lysates and are expressed in ng/mL ± SEM (n = 3).

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It is known that decreased levels of cellular glutathione culminate in oxidative stress. Hence, we measured total cellular glutathione levels in primary cortical cultures treated with vehicle, 30 µm tBHQ or 30 µm DEM in the presence and absence of 2 mm GSHEE. Such measurements would allow us to associate the activation of the ARE with oxidative stress in the primary cortical cultures. Total cellular GSH levels in cultures treated with 30 µm DEM were reduced by 72% from that of vehicle (Fig. 4b), whereas GSH levels in cultures pre-treated with 2.0 mm GSHEE followed by 30 µm DEM were not different from cultures treated with vehicle. Contrary to DEM, 30 µm tBHQ did not reduce GSH compared to vehicle treated cultures with or without GSHEE pre-treatment (Fig. 4b).

Inhibition of hPAP Activity by LY294002 in primary neuronal cultures treated with tBHQ

More recently, our lab has shown that PI3-kinase regulates the activation of the ARE in IMR-32 human neuroblastoma cells (Lee et al. 2001b). To determine if this was consistent in primary cultures, primary cortical cultures derived from the ARE-hPAP reporter mice were pre-treated (20 min) with vehicle or increasing doses of LY 294002, a selective PI3-kinase inhibitor. Cultures were then treated with 30 µm tBHQ and lysates harvested at 24 and 72 h. At 24 h, 15 µm and 30 µm LY 294002 completely blocked tBHQ activation of the ARE and relative hPAP activities were at or near activity observed in the vehicle-treated samples (Fig. 5, 24h). At 72 h, LY 294002 in a dose dependent fashion inhibited the activation of ARE from 26-fold down to 10-fold (Fig. 5, 72h). Cell viability of the cultures was determined by the MTS cytotoxicity assay (Promega) and in all treatment groups no toxicity was observed (data not shown).

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Figure 5. Expression of hPAP in primary cortical cultures in the presence and absence of LY 294002 and tBHQ. Primary cortical tissue from E16 embryos was harvested and cultured in 96-well plate format as described Experiment Procedures. Cultures were pre-treated for 20 min with increasing doses of LY 294002 followed by treatment with vehicle [0.03% (v/v) ethanol] or tBHQ (30 µm) for 24 h (a) or 72 h (b). Cell lysates were analyzed for hPAP activity and values represent the mean ± SEM (n = 5). *Significantly different than corresponding vehicle-treated sample (p < 0.05).

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RT-PCR of total RNA isolated from primary neuronal cultures from ARE-hPAP reporter mice

Previous studies described in this report demonstrated the activation of the ARE through reporter gene assays and histochemistry. In support of these data, we isolated total RNA from primary cortical cultures derived from the ARE-hPAP transgenic mice and analyzed levels of gene expression for hPAP and NQO1 using RT-PCR. In addition, we amplified total RNA for GCL regulatory subunit (GCLR), a gene also known to be mediated by the ARE (Moinova and Mulchay 1988) and Nrf-2, the transcription factor reported to regulate the ARE (Venugopal and Jaiswal 1996; Lee et al. 2001a). Cultures were treated with vehicle [0.03% (v/v) ethanol] or 30 µm tBHQ in the presence or absence of 30 µm LY 294002 and 24 h later total RNA was isolated. As shown in Fig. 6, tBHQ-treated cultures had increased mRNA above basal levels for hPAP, NQO1, and the GCLR. All were inhibited by the presence of LY 294002. Interestingly, although tBHQ did not influence Nrf2 gene expression, LY 294002 appeared to decrease mRNA levels for Nrf2. β-actin gene expression was used for amplification control and was not changed in any treatment groups.

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Figure 6. Expression of ARE-mediated genes in primary cortical cultures. Cultures were treated for 24 h after which RNA was isolated and subjected to RT-PCR for hPAP, NQO1, GCLR, Nrf-2 and β-actin. The resulting products formed were analyzed by agarose gel electrophoresis. Lanes 1–4 represent RNA isolated from primary neuronal cultures treated with the following: lane 1, treated with vehicle [0.3% (v/v) EtOH; − 20 min] and vehicle [0.03% (v/v) EtOH; 24 h]. Lane 2, treated with LY 294002 (30 µm; − 20 min) and vehicle [0.03% (v/v) EtOH; 24 h]. Lane 3, treated with vehicle [0.3% (v/v) EtOH; − 20 min] and tBHQ (30 µm; 24 h). Lane 4, treated with LY 294002 (30 µm; − 20 min) and tBHQ (30 µm; 24 h).

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Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We have successfully created mice that carry in their genome a stable hPAP reporter gene that is basally and transcriptionally controlled by a segment of the rat NQO1 promoter containing the core ARE sequence. This was demonstrated by the dose-dependent activation of hPAP by tBHQ in primary cortical cultures derived from the ARE-hPAP mice. In primary cortical cultures treated with tBHQ for 72 h, cell-specific activation of hPAP was identified in astrocytes and neurons. Histochemical staining for NQO1 followed in a similar fashion. A selective PI3-kinase inhibitor, LY 294002, blocked the activation of the ARE by tBHQ suggesting this activation was mediated through a PI3-kinase dependent mechanism involving Nrf2.

Recently published data from our lab demonstrated that the activation of the ARE by tBHQ and DEM in IMR-32 neuroblastoma cells was mediated through the transcription factor Nrf2 (Lee et al. 2001a). In addition, activation of the ARE by tBHQ was independent of oxidative stress, while activation of the ARE by DEM was dependent on oxidative stress. This implies that tBHQ and DEM can activate the ARE by different signaling pathways that converge on Nrf2. Similarly, in primary cultures derived from the ARE-hPAP reporter mice, pre-treatment with GSHEE blocked activation of the ARE by DEM but not tBHQ, suggesting the activation of the ARE by tBHQ was independent of oxidative stress, whereas activation of the ARE by DEM was a result of oxidative stress. Furthermore, total cellular GSH levels from primary cultures treated with DEM showed a marked decrease compared to that of vehicle which was reversed by pre-treatment with GSHEE. GSH levels in primary cultures treated with tBHQ were unaffected. However, to truly confirm these observations, continuing experiments in our laboratory are focused on measuring levels of oxidative stress biomarkers such as 8-oxo-7,8-dihydro-2′-deoxyguanosine, a marker of DNA damage, and 4-hydroxynonenal, a lipoxidation product.

We theorize that the activation of the ARE in neurons is also through a Nrf2-dependent mechanism. To test this theory, we have begun to cross breed our ARE-hPAP reporter mice with the Nrf2 knock-out mouse (Chan and Kan 1999) to study the effects of the absence of Nrf-2 on basal and inducible activation of the ARE in primary cortical cultures and in vivo. Work in our lab has also shown that activation of the ARE in neuroblastoma cells is regulated by PI3-kinase (Lee et al. 2001b). Likewise, in primary cortical cultures derived from the ARE-hPAP mice, LY 294002, in a dose-dependent manner, inhibited tBHQ activation of the ARE, although not completely at 72 h. This incomplete inhibition may suggest an alternative ARE-signaling pathway that does not depend on PI-3 kinase, or perhaps that the half-life of tBHQ is longer than that of LY 294002. Thus, as evident at the 24-h time point, LY 294002 would be a more effective inhibitor of PI-3 kinase than at 72 h. RT-PCR of RNA isolated from these cultures treated with tBHQ showed an induction of NQO1 and GCLR gene expression that was blocked by LY 294002. Interestingly, the basal gene expression of Nrf2 was inhibited by LY 294002. This loss of Nrf2 expression could significantly contribute to the inhibitory effects of LY 294002 on ARE-driven genes. It is possible that treatment with LY 294002 does not directly block tBHQ-mediated signaling, but indirectly modulates the level of Nrf2 that is required for activation of the ARE. If PI3-kinase is responsible for maintaining expression of Nrf2, then inhibition of this kinase would result in reduced levels of Nrf2 and loss of inducibility by tBHQ. In either case, it would appear that PI-3 kinase plays a major role in ARE-driven gene expression in primary cortical cultures.

Chan and Kan (1999) demonstrated the importance of the transcription factor Nrf2 when Nrf2 knockout mice fed BHA showed extreme pulmonary toxicity. Gene expression of NQO1, HO1 and CGLR were significantly reduced in the lungs of these animals suggesting these ARE-containing genes, regulated by Nrf2, play a critical role in protection from oxidative stress. Preliminary data using oligonucleotide microarrays and RT-PCR to compare the expression profiles of control neurons from Nrf2–/– and Nrf2+/+ littermates, indicate that NQO1, CGLR, GCLC and HO-1 mRNA levels are significantly reduced in the Nrf2–/– cultures making them more susceptible to oxidative stress (J. M. Lee and J.A. Johnson, unpublished observation). These findings support our hypothesis that the coordinate induction of the ARE-containing genes may confer protection against neuronal cell death.

Activation of the ARE by tBHQ was observed in neurons and astrocytes. Neurons that stained positive were in close proximity to astrocytes. Perhaps the astrocytes in these cultures are secreting obligatory factors required for activation of the ARE in neurons. This observation was supported by the increased number of neurons staining positive for hPAP activity in cultures maintained in the absence of AraC, a mixed culture system of astrocytes and neurons (Fig. 2b), compared with cultures maintained in the presence of AraC which are predominately neurons (Fig. 2d). This is the first report indicating that the ARE and NQO1 can be activated and expressed, respectively, in cultured neurons. The data suggest that although post-mitotic-differentiated neurons in human and rodent brain are consistently negative for NQO1, they may have the capacity to reverse this differentiation imposed repression of the ARE and genes controlled by this enhancer element.

In contrast to the data presented here, transient transfection of ARE reporter gene constructs in rat primary cortical cultures that were treated with DMF for 24 h, showed preferential activation of the ARE in astrocytes, but not in neurons (Murphy et al. 2001). We attribute the inability of neurons to express the ARE in this system to a shorter (24 h vs. 72 h) exposure of the cultures to the ARE activator, DMF. The longer exposure may afford the non-neuronal cell population the time needed to manufacture and secrete factors required by neurons to activate the ARE. In addition, Laxton et al. (2001) demonstrated that in rats given a unilateral focal cerebral infarct, NQO1 activity was significantly increased in astrocytes localized to the cortex and subcortical areas as compared with the non-ischemic side. NQO1 activity in neurons was not detected. In this system, ischemia resulted in an acute oxidative insult and rapid neuronal cell death. It would therefore be unlikely to observe NQO1 activity in neurons that are dying or in the process of apoptosis.

In support of the ARE activation in neurons, Wang et al. (2000) reported that in brains from AD patients, NQO1 activity was observed in hippocampal neurons and was undetectable in the same neuronal population in healthy controls. It was hypothesized that increased NQO1 was a result of the activation of the ARE in response to the reactive oxygen species generated by the accumulation of amyloid-beta protein (Aβ). However, as stated earlier, the stable overexpression of NQO1 in a neuroblastoma cell line was not sufficient to block oxidative stress-induced cell death (Murphy et al. 1991). This would imply that the coordinate activation of ARE-driven genes may be required for neuroprotection. In the AD brain, all the surviving neurons in close proximity to the pathology were NQO1-positive, while only a small fraction were positive for phospho-tau. The question remains as to the effect of increased ARE-driven gene expression on Aβ toxicity. Future experiments with the ARE-hPAP reporter mice therefore will aim to correlate the activation of the ARE in neurons and glia with neuroprotection against Aβ toxicity in primary cortical cultures.

Clearly, screening for ARE-activating compounds could prove to be important in the role of drug discovery. The ARE-hPAP reporter mice have the potential to be used for such purposes to determine the cell-specific activation of the ARE by various drugs. The ARE-hPAP reporter mice could also be used as an extremely sensitive model for the determination of chemicals that cause oxidative stress. Lastly, the identification of the signaling pathway in the activation of the ARE has not fully been elucidated. Studies using primary cultures or tissue from the ARE-hPAP reporter mice in correlation with hPAP activity can only further our knowledge of the ARE signaling pathway.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by the National Institute of Environmental Health Science Grants, ES08089 and ES10042, and the Burroughs Wellcome New Investigator in Toxicological Sciences Award awarded to JAJ. The National Institute of Environmental Health Science Grant, ES05704, awarded to GKA also supported this work.

References

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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