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Am J Physiol Cell Physiol 290: C95-C103, 2006. First published August 24, 2005; doi:10.1152/ajpcell.00388.2005
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GROWTH, DIFFERENTIATION, AND APOPTOSIS

Apolipoprotein A-IV attenuates oxidant-induced apoptosis in mitotic competent, undifferentiated cells by modulating intracellular glutathione redox balance

Heather L. Spaulding,1 Fumito Saijo,1 Richard H. Turnage,1 J. Steven Alexander,2 Tak Yee Aw,2 and Theodore J. Kalogeris1,2

Departments of 1Surgery and 2Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana

Submitted 1 August 2005 ; accepted in final form 18 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Oxidant-mediated modulation of the intracellular redox state affects the apoptotic cascade by altering the balance between cellular signals for survival and suicide. Apolipoprotein A-IV (Apo A-IV) is known to possess antioxidant-like activity. In the present study, we tested 1) whether Apo A-IV could influence redox-dependent apoptosis and, if so, 2) whether such an effect could be mediated by modulation of intracellular redox balance. Mitotic competent, undifferentiated PC-12 cells were incubated with either tert-butyl hydroperoxide (TBH) or diamide with or without preincubation with human Apo A-IV. Apo A-IV significantly decreased apoptosis produced by both TBH and diamide, and washout of A-IV before incubation with TBH and diamide did not eliminate its protective effect. Apo A-I had no such protective effect. The Apo A-IV effect was not blocked by D,L-buthionine-[S,R]-sulfoximine, but it was reversed by both dehydroisoandrosterone and transfection with an antisense oligodeoxynucleotide to glucose-6-phosphate dehydrogenase (G6PD). Apo A-IV abolished the transient, oxidant-induced rise in glutathione disulfide (GSSG) and cellular redox imbalance previously shown to initiate the apoptotic cascade. Apo A-IV had no effect on GSSG reductase activity, but it stimulated G6PD activity 10-fold. These results suggest a novel role for Apo A-IV in the regulation of intracellular glutathione redox balance and the modulation of redox-dependent apoptosis via stimulation of G6PD activity.

tert-butyl hydroperoxide; diamide; dehydroisoandrosterone; glucose-6-phosphate dehydrogenase; antisense

APOLIPOPROTEIN A-IV (Apo A-IV) was first described >25 years ago as a component of intestinally secreted, triglyceride-rich lipoproteins (27). Under basal conditions, Apo A-IV is secreted from the small intestine; its synthesis and secretion dramatically increase in response to a fatty meal (1, 15, 16). Upon secretion, Apo A-IV appears on lymph chylomicrons; during subsequent plasma passage and metabolism of chylomicrons, ~25% of circulating Apo A-IV is transferred to the HDL fraction and the remaining 75% is found in the lipoprotein-free fraction of plasma (13, 19). Apo A-IV has several proposed physiological roles, including lipid transport, lipoprotein metabolism, and control of food intake and gastric function (17). Most recently, the findings in studies of transgenic mice have supported a role for Apo A-IV in protection against atherosclerosis (6, 9, 12, 21). Recent findings in in vitro studies have supported an antioxidant action of Apo A-IV (11, 25). This latter activity has been proposed (21, 25) to explain its antiatherogenic action as an alternative or complementary mechanism to A-IV's presumed ability to promote cellular lipid efflux (12). A clear mechanistic link between Apo A-IV's antioxidant activity and its ability to protect has not been established. However, because atherosclerosis is now recognized as a chronic inflammatory disease (26) and because antioxidant compounds are protective in experimental models of inflammation, demonstration of Apo A-IV-mediated anti-inflammatory activity could provide a crucial link between its antioxidant activity and its ability to protect. Ongoing work in our laboratory (32) points to a general anti-inflammatory action of Apo A-IV; thus its ability to prevent atherogenesis may be one instance of a much broader protective role for this protein. How such a role might manifest at the cellular level is unknown.

Recent studies by Pias and Aw (22), who used undifferentiated pheochromocytoma (PC-12) cells challenged with tert-butyl hydroperoxide (TBH), demonstrated a linkage between oxidant stress, intracellular glutathione-glutathione disulfide (GSH-GSSG) redox imbalance, and the mitochondrial apoptotic pathway (22). Further studies using the cell-permeant thiol oxidant diamide showed that GSH redox imbalance per se was sufficient to trigger the apoptotic cascade independently of reactive oxygen species (ROS) (23). Kinetic studies showed that a transient redox imbalance occurring within 30 min of the initial ROS challenge resulted in a significant increase (in temporal order) in Bax activation and Bcl-2 downregulation, mitochondrial cytochrome c release, caspase activation, and eventually apoptosis that was reversible only by N-acetyl cysteine (NAC, a GSH precursor) or exogenous GSH when these were added either before or simultaneously with oxidant challenge (22, 23). Thus oxidant-induced apoptosis is dependent on a rapid and transient imbalance in intracellular redox. The precise linkage between this transient redox imbalance and triggering of the apoptotic cascade has not been elucidated yet; however, it is clear that attempts to modulate ROS-induced apoptosis at the level of intracellular redox must take into account a narrow temporal window within which prevention or reversal of redox imbalance must occur.

The studies of Pias and Aw (22, 23) demonstrated that the PC-12 system is a valid and useful model for studying oxidant-induced, redox-dependent cell responses. Thus, as a first step in examining the potential for Apo A-IV as a cytoprotective protein, we tested its effects on the well-characterized apoptotic response to oxidants in the PC-12 model system. We sought answers to the following specific questions: 1) Does Apo A-IV modulate the apoptotic response in cells exposed to a model hydroperoxide (TBH)? 2) Because the apoptotic response to TBH depends on production of both ROS and intracellular redox imbalance, can Apo A-IV similarly protect against redox-dependent, ROS-independent apoptosis? 3) Can Apo A-IV itself modulate intracellular redox? The results of these studies indicate a novel and heretofore unsuspected role of Apo A-IV in modulating intracellular redox balance.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. PC-12 cells were cultured in DMEM containing 10% heat-inactivated FBS, 5% heat-inactivated horse serum, penicillin (100 U/ml), streptomycin (100 µg/ml), amphotericin (25 µg/ml), and glutamine (2 mM). The cultures were grown and maintained in a humidified 5% CO2 atmosphere at 37°C. The medium was changed every 2–5 days, and cells were passaged every 5 days.

Recombinant apolipoprotein A-IV. We produced recombinant human Apo A-IV (rhA-IV) using the Apo A-IV expression vector pL2102-hA-IV, which is a modified version of pL1867-A-IV previously described by Duverger et al. (8). This construct encodes the full-length protein-coding portion of the Apo A-IV gene, with a 6x histidine tag to facilitate purification. rhA-IV from this construct was previously demonstrated to be physicochemically and functionally similar to native human Apo A-IV purified from plasma (8). rhA-IV was produced in BL21(DE3)pLysS Competent Cells (Promega, Madison, WI) after transformation with pL2102-hA-IV. After overnight culture, cells were diluted 1:4 with fresh medium, allowed to grow to optical density (OD)610 0.5, and then induced for 1–2 h with 1 mM isopropyl-D-thiogalactoside (IPTG). Cells were harvested, washed, and then lysed in 20 mM PBS (in mM: 19.7 NaH2PO4, 0.26 Na2HPO4, and 500 NaCl, pH 7.8) by freeze-thawing followed by sonication. Bacterial lysates were incubated in batchwise fashion with Ni2+-charged Sepharose resin (Pro-Bond resin; Invitrogen, Carlsbad, CA) and then poured into an 18 x 2-cm column. The column was washed extensively with 20 mM PBS, followed by 20 mM PBS with 40 mM imidazole; completeness of washing was monitored via continuous measurement of OD280 and by performing SDS-PAGE on selected fractions. The rhA-IV was eluted from the column free of contaminating bacterial proteins using 20 mM PBS and 150 mM imidazole. Eluted rhA-IV was dialyzed exhaustively against PBS (pH 7.4), concentrated against polyethylene glycol (PEG) 8000, filter sterilized, and then stored in small aliquots at –80°C until needed. Freshly thawed rhA-IV was used for all experiments.

Native apolipoprotein A-IV. Human Apo A-IV was isolated from lipoprotein-depleted serum (LPDS) using a modification (34) of the method of Weinberg and Scanu (33). All procedures using human subjects were reviewed and approved by the Institutional Review Board of Louisiana State University Health Sciences Center. Serum was obtained from healthy, nonfasting volunteers and raised to a density of 1.25 g/ml by the addition of solid KBr and then centrifuged at 38,000 rpm for 48 h at 10°C in a Beckman SW-41 rotor. Floating lipoproteins were aspirated after tube slicing, and the lipoprotein-free subnatant was dialyzed exhaustively against 0.05 M K+ phosphate buffer (pH 7.4) containing 0.05% EDTA at 4°C. The LPDS was then incubated with 10% intralipid fat emulsion in 4 M NaCl and 50 mM phosphate, pH 7.4, at 37°C. The emulsion-LPDS mixture was centrifuged at 28,000 rpm for 35 min at 4°C in a Beckman SW-28 rotor, and the floating cream layer was removed and then delipidated in 50 volumes of 3:1 (vol/vol) diethyl ether/ethanol. The protein precipitate was collected, washed in diethyl ether, and dried under a vacuum. The protein was solubilized in 50 mM Tris, pH 8.2, and urea at 4°C. The protein fraction (30 mg) was then applied to a 40 x 1.6-cm column of DEAE-cellulose (DE52; Whatman, Florham Park, NJ) equilibrated with 50 mM Tris, pH 8.2, at 4°C. The column was washed with 200 ml of equilibration buffer and then eluted with an 800-ml gradient from 50–90 mM Tris. The column effluent was monitored continuously at 280 nm, and 4-ml fractions were collected. Apo A-IV was eluted at 74 mM Tris. During establishment of this method in our laboratory, Apo A-IV identity was confirmed using direct sequence analysis; for routine purification, fractions were assayed for Apo A-IV using immunoblot analysis and checked for purity using SDS-PAGE on 10% gels. Fractions containing Apo A-IV were dialyzed exhaustively against HBSS (pH 7.4), concentrated in the dialysis cassette using PEG 8000, and then filter sterilized, assayed for protein concentration, and either stored at –80°C or lyophilized and stored at 4°C until use.

Effect of Apo A-IV on oxidant-induced apoptosis. Twenty-four hours before each experiment, cells were seeded at a density of 105 cells/ml on 12-mm-diameter circular glass coverslips. Cells were incubated in culture medium for 1 h with or without rhA-IV or native Apo A-IV at a concentration of 25, 50, 100, 200, 400, or 800 µg/ml. This concentration range brackets plasma Apo A-IV levels, which, depending on the species, are from 50 to 200 µg/ml. To test the specificity of Apo A-IV's effects, we also tested recombinant Apo A-I (1,280 µg/ml). Cells were then incubated with TBH at 100 µM administered as a single bolus to the cells 24 h before being fixed and stained. Similar studies were conducted using the cell-permeant thiol oxidant diamide (100 µM). All apoptosis experiments were conducted in either triplicate or quadruplicate for each treatment. Twenty-four hours after treatment, the cells were washed with PBS and fixed for 15 min in ice-cold 4% paraformaldehyde. Cells were then washed with PBS and fixed for at least 1 h at –20°C with ice-cold 70% ethanol. Coverslips were mounted on glass slides using Vectashield mounting medium containing 4-6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). Cells were viewed and counted at x40 magnification using an Olympus Bx50 fluorescence microscope. On each slide, at least 200 apoptotic and total cells were counted in six random fields of view. Cells were judged to be apoptotic on the basis of the presence of clearly observed chromatin condensation, nuclear fragmentation, and apoptotic bodies (22). In separate preliminary studies, the effect of TBH on cellular release of lactate dehydrogenase (LDH) was measured using a commercially available cell toxicity kit (Sigma, St. Louis, MO) to compare the LDH released in response to a given treatment with that measured in cells treated with 0.1% Triton X-100. After 24 h of exposure to TBH (100 µM), measurement of LDH release indicated that ~2–3% cell death was due to necrosis, a finding that is not significantly different from that for control cells that were not treated with TBH. Diamide produced similarly low levels of LDH release (1.5–2%).

In separate experiments, we tested whether Apo A-IV's antiapoptotic effect is due to modulation of GSH redox balance per se or to an effect on a downstream event by comparing the effect of varying the temporal order of exposure of cells to Apo A-IV vs. oxidant. Cells were treated with 200 µg/ml rhA-IV either 1 h before or 30 min after exposure to TBH or diamide. Apoptosis was then quantified 24 h after the start of oxidant treatment.

Because previous studies showed that eventual apoptosis requires a transient imbalance in GSH redox occurring within a brief temporal window after treatment with oxidant, we examined whether the antiapoptotic effect of Apo A-IV was dependent on the continued presence of Apo A-IV in the cell medium upon treatment. After treating cells for 1 h with 200 µg/ml Apo A-IV, we either washed out the rhA-IV with three changes of media before adding TBH or diamide or added the oxidants to rhA-IV-containing medium. Both groups were then treated with either TBH or diamide at 100 µM, and apoptosis was measured as described above.

In a fourth set of studies, we tested Apo A-IV's antiapoptotic activity during inhibition of key enzymes mediating either GSH synthesis or GSH-GSSG redox cycling. Cells were preincubated for 1 h with either 1 mM D,L-buthionine-[S,R]-sulfoximine (BSO, GSH synthase inhibitor) or 200 µM dehydroepiandrosterone [DHEA, a glucose-6-phosphate dehydrogenase (G6PD) inhibitor] (23, 29) in the presence or absence of 800 µg/ml rhA-IV. They were then exposed to TBH (100 µM) for 24 h and assessed for apoptotic response.

To test more specifically the role of G6PD in the antiapoptotic effect of Apo A-IV, we used antisense methods originally described by Leopold et al. (20). PC-12 cells were transfected with an antisense phosphorothioate oligodeoxynucleotide (Invitrogen) to G6PD mRNA (G6PD-A, 5'-AGGUCACCCGAUGCACCCAUGAUGA-3') or a control, scrambled oligo, G6PD-Sc, 5'-CAGAUAGAAUGCACUGGUCCGCCCC-3', using oligofectamine (Invitrogen). After 48 h, they were treated with TBH with or without pretreatment with rhA-IV as described above and then assayed for either G6PD expression using Western blot analysis (see below), G6PD activity (see below), or TBH-induced apoptosis.

Measurement of activity of G6PD and GSSG reductase. We tested whether Apo A-IV could modulate activity of the two major enzymes responsible for regenerating GSH from GSSG: G6PD and GSSG reductase. Cells were seeded onto 100-mm dishes (4 x 106 cells/dish), and 24 h later they were incubated with 800 µg/ml Apo A-IV for 24 h. Cells were then harvested by being scraped into PBS and lysed using sonication. Lysates were assayed for G6PD (24) and GSSG reductase (14) as described previously. Similar procedures for measuring G6PD activity were used in cells treated with G6PD antisense or scrambled control oligodeoxynucleotides.

Western blot analysis of cellular G6PD protein. PC-12 cells plated in 60- or 100-mm dishes were treated with either rhA-IV (800 µg/ml) for graded periods up to 1 h or G6PD antisense or scrambled control oligodeoxynucleotides. They were then washed with ice-cold PBS and lysed using sonication in 50 mM Tris (pH 8.0), 150 mM NaCl, 0.5% SDS, 1% Triton X-100, 1 µg/ml aprotinin, and 100 µg/ml PMSF. Homogenates were centrifuged at 2,000 g for 10 min to remove cellular debris, and protein content was measured using the Bradford assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of supernatant protein (30 µg) were then subjected to SDS-PAGE, followed by Western blotting onto nitrocellulose membranes. The blots were probed with goat anti-G6PD antiserum (USBiological, Swampscott, MA), followed by horseradish peroxidase-coupled rabbit anti-goat IgG. Blots were developed using an ECL reagent system (Amersham Biosciences, Piscataway, NJ) and then scanned for optical density to determine relative levels of G6PD between treatments.

Effect of Apo A-IV on TBH-induced shift in redox balance. The apoptotic response to both TBH and diamide has been shown to be preceded by a transient shift in GSH-GSSG balance toward GSSG, i.e., toward a more oxidized state (22, 23). In this experiment, we tested the ability of Apo A-IV to modulate the oxidant-induced shift in GSH redox using a HPLC method described previously (22). Twenty-four hours before the experiment cells were grown at a density of 106 cells/ml on P-100 culture plates. One hour before the experiment culture medium was replaced with 4 ml of fresh serum-free medium with or without 800 µg/ml Apo A-IV. The cells were then incubated with 100 µM TBH for 0, 5, 15, and 30 min (triplicate plates run at each time point). At the end of the experimental period, cells were washed twice with PBS and scraped from the dish into ice-cold 5% TCA. Suspensions were centrifuged to precipitate the TCA-insoluble proteins. TCA supernatant was derivatized with 6 mM iodoacetic acid and 1% 2,4-dinitrophenyl fluorobenzene to yield the S-carboxymethyl and 2,4-dinitrophenyl derivatives of GSH and GSSG. GSH and GSSG derivatives were separated by performing HPLC on a 250 x 4.6-mm Alltech Lichrosorb NH2-terminal 10-µm column. Cellular GSH and GSSG contents were quantified by comparing them with standards derivatized in the same manner.

Statistical analysis. Data were analyzed using either an unpaired t-test or one-way ANOVA with multiple comparison testing using the Bonferroni correction. Differences were considered significant at P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Apo A-IV significantly decreases apoptosis in PC-12 cells elicited by TBH and diamide. In all experiments, the proportion of apoptotic cells under control conditions (i.e., in the absence of oxidant challenge) ranged from 6% to 9%. Apo A-IV alone had no effect, because the apoptotic response in PC-12 cells incubated with rhA-IV (800 µg/ml) was within the aforementioned control range (7.4–9.2%) (Fig. 1).



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  		Fig. 1. Concentration-dependent effects of apolipoprotein A-IV (Apo A-IV) on oxidant-induced apoptosis in pheochromocytoma (PC-12) cells. PC-12 cells on glass coverslips were incubated for 1 h with or without recombinant human Apo A-IV (rhA-IV) and then were treated for 24 h with 100 µM tert-butyl hydroperoxide (TBH; A) or 100 µM diamide (B). Apoptosis was determined by 4–6-diamidino-2-phenylindole (DAPI) staining as described in MATERIALS AND METHODS and was expressed as the percentage of cells exhibiting apoptotic morphology (i.e., nuclear fragmentation and apoptotic bodies). Values are means ± SE from 4 separate experiments. abcP < 0.05, bars labeled with different letters are significantly different from one another.

 
TBH produced a 2.4-fold increase in the fraction of cells that exhibited apoptotic morphology (Fig. 1A). Preincubation of cells with rhA-IV produced significant, dose-dependent decreases in the percentage of apoptotic cells, with 200 and 800 µg/ml A-IV decreasing TBH-induced apoptosis by 30% and 45%, respectively (Fig. 1A). A similar pattern was observed in the diamide studies (Fig. 1B). Diamide produced a twofold increase in apoptosis that was significantly decreased by 29% and 43% using rhA-IV at 200 and 800 µg/ml, respectively.

Apo A-IV prevents TBH-induced increase in GSSG and decrease in GSH-GSSG. We measured the kinetics of TBH-induced changes in cellular GSH and GSSG at different times after treatment of cells with 100 µM TBH with or without rhA-IV (800 µg/ml) (Fig. 2). Figure 2A shows that TBH produced an initial decrease in GSH levels at 5 min (not significant) and a further decrease to a plateau by 15 min (P < 0.05). Preincubation with rhA-IV had no effect on the GSH response. TBH produced a transient increase in GSSG that peaked at 5 min and was >20-fold higher than control GSSG levels (P < 0.001) (Fig. 2B). GSSG returned to a level not significantly different from control by 15 min. This finding is consistent with an initial rapid oxidation of GSH to GSSG, followed by subsequent reduction of GSSG via GSSG reductase. Pretreatment with rhA-IV completely abolished the transient rise in GSSG at 5 min (P < 0.001) (Fig. 2B). Because of the transient increase in GSSG, cellular redox (i.e., GSH-to-GSSG ratio) showed a transient decrease to <10% of control in response to TBH (P < 0.001) (Fig. 2C). Pretreatment with rhA-IV significantly reversed this decline (P < 0.001 vs. TBH alone). Thus pretreatment with rhA-IV, by abolishing the transient increase in GSSG, prevented the cellular redox imbalance caused by TBH and maintained GSH-GSSG at control levels.



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  		Fig. 2. Graphs showing the kinetics of change in intracellular glutathione (GSH) and glutathione disulfide (GSSG) induced by TBH or TBH with rhA-IV. PC-12 cells (1 x 106 cells) in 35-mm culture dishes were treated with 100 µM TBH in the presence or absence of rhA-IV (800 µg/ml) for up to 30 min. At designated times, samples were collected and derivatized for analysis of GSH and GSSG by performing HPLC as described in MATERIALS AND METHODS. A: GSH. B: GSSG. C: GSH-to-GSSG ratio. Cellular concentrations of GSH and GSSG are expressed as nM/106 cells and are presented as means ± SE from 7 separate experiments (control) and 4 separate experiments each for both treatments (TBH or TBH + rhA-IV). *P < 0.001 vs. control. #P < 0.001 vs. TBH alone.

 
To confirm the lack of effect of Apo A-IV on cellular GSH levels, additional control studies were performed in which GSH was measured in response to Apo A-IV, the GSH synthesis inhibitor BSO, or Apo A-IV and BSO together. GSH in control cells was 25 ± 0.53 nmol/mg of protein, but incubation with rhA-IV had no effect on GSH (23.5 ± 5.2 nmol/mg of protein). In response to BSO, cellular GSH levels decreased to 15 ± 1.95 nmol/mg of protein (P < 0.05) but pretreatment of cells with rhA-IV did not modulate BSO's effect (14.4 ± 0.6 nmol/mg of protein).

Apo A-IV attenuates oxidant-induced apoptosis within a narrow temporal window. Pretreatment of cells with rhA-IV for 1 h before exposure to either TBH or diamide attenuated oxidant-induced apoptosis by 30% (P < 0.01) (Fig. 3). However, if rhA-IV treatment was delayed until 30 min after the start of incubation with oxidant, A-IV's antiapoptotic effect was completely abolished. This finding is consistent with the antiapoptotic effect of Apo A-IV being mediated by its prevention of the transient, oxidant-induced redox imbalance (Fig. 2).



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  		Fig. 3. Bar graphs showing effect of order of treatment of cells with rhA-IV in relation to exposure to oxidants. PC-12 cells on glass coverslips were incubated with or without TBH or diamide (100 µM) for 24 h. Cells were either preincubated with rhA-IV (200 µg/ml) for 1 h before the start of oxidant exposure (pretreat) or incubated with rhA-IV beginning 30 min after the start of oxidant exposure (post-treat). Apoptosis was quantified as described in the Fig. 1 legend. Values are means ± SE from 4 separate experiments. abcP < 0.01, letters denote significant differences.

 
Native and recombinant Apo A-IV are similarly effective in decreasing TBH-induced apoptosis, but Apo A-I is ineffective. To test the specificity of Apo A-IV's protective effect, we compared the effects of rhA-IV with native A-IV isolated from plasma (both at 800 µg/ml). Both produced a significant 37% decrease in apoptosis. In contrast, treatment with the related protein, Apo A-I, at an equivalent concentration (497 µg/ml) did not block TBH-induced apoptosis and in fact produced an apoptotic response that significantly exceeded that observed in response to TBH alone (Fig. 4).



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  		Fig. 4. Bar graphs comparing the effect of rhApo A-IV on TBH-induced apoptosis with that of native Apo A-IV purified from human serum (described in MATERIALS AND METHODS) and native human Apo A-I. PC-12 cells were preincubated with 800 µg/ml Apo A-IV or an equimolar concentration of Apo A-I (498 µg/ml) for 1 h, followed by 24 h incubation with TBH (100 µM). Apoptosis was quantified as described in the Fig. 1 legend. Values are means ± SE from 4 separate experiments. abcP < 0.01, letters denote significant differences.

 
Preincubation of cells with rhA-IV followed by washout before exposure to oxidants does not block A-IV's antiapoptotic effect. We preincubated PC-12 cells with rhA-IV at 200 µg/ml for 1 h. rhA-IV in the media was thoroughly washed out by performing several media changes. Extensive Western blot analysis showed that the above protocol completely removed Apo A-IV from the media. After washout, the cells were incubated with TBH or diamide in fresh media. Washout of rhA-IV did not attenuate its antiapoptotic effect. Significant 30% and 46% decreases in TBH- and diamide-stimulated apoptosis, respectively, were recorded (Fig. 5).



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  		Fig. 5. Bar graphs showing the effect of washout of rhA-IV from culture medium oxidant-induced apoptosis. Cells were incubated with rhA-IV (200 µg/ml) for 1 h, and then the Apo A-IV-containing media was removed and cells were washed three times with non-Apo A-IV-containing medium and incubated with fresh medium containing only TBH (100 µM) or diamide (100 µM). Values are means ± SE from 4 separate experiments. abcP < 0.05, letters denote significant differences.

 
Inhibition of de novo synthesis of GSH does not block Apo A-IV's antiapoptotic effect. Inhibition of de novo GSH synthesis using BSO did not reverse the antiapoptotic effect of Apo A-IV, which effected a significant 38% decrease in TBH-induced apoptosis (Fig. 6). These results are consistent with the lack of effect of A-IV on the cellular synthesis of GSH (Fig. 2A).



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  		Fig. 6. Graphs showing the effect of rhA-IV on TBH-induced apoptosis in the presence of inhibition of GSH synthesis by the GSH synthase inhibitorD,L-buthionine-[S,R]-sulfoximine (BSO). PC-12 cells were preincubated with BSO (1 mM) in the presence or absence of rhA-IV (800 µg/ml) for 1 h. They were then incubated for a further 24 h with TBH (100 µM) and assessed for apoptotic response. Values are means ± SE from 4 separate experiments. abcP < 0.05, letters denote significant differences.

 
Apo A-IV stimulates G6PD activity. The other key enzymes involved in control of GSH-GSSG redox are GSSG reductase and G6PD. Cells incubated with rhA-IV for 1 h had little effect on activity of GSSG reductase but caused a 10-fold increase in G6PD activity (Table 1). This increase was almost totally abolished in cells coincubated with the G6PD inhibitor DHEA (6.3 ± 1.6 nmol NADPH·min–1·mg–1).


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  		Table 1. Effect of apolipoprotein A-IV on the activity of GSSG reductase and G6PD

 
G6PD activity is required for Apo A-IV-mediated protection against TBH-induced apoptosis. Because of the striking effect of Apo A-IV on G6PD activity, we next tested whether G6PD activity is necessary for Apo A-IV's antiapoptotic effect using the inhibitor DHEA (Fig. 7). Whereas Apo A-IV decreased TBH-induced apoptosis by almost 40%, cotreatment of cells with DHEA completely abolished A-IV's effect. When treated with DHEA alone, cells exhibited a significant 76% increase in apoptosis and pretreatment with rhA-IV had no effect on this increase (Fig. 7). These results, together with the measurements of G6PD activity described above, argue against the possibility that Apo A-IV might somehow bind to or otherwise directly inactivate DHEA and indicate that Apo A-IV's antiapoptotic effect is mediated through stimulation of G6PD.



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  		Fig. 7. Graphs showing the effect of rhA-IV on TBH-induced apoptosis during inhibition of glucose-6-phosphate dehydrogenase (G6PD) activity by the a G6PD inhibitor dehydroepiandrosterone (DHEA). PC-12 cells were preincubated with DHEA (200 µM) in the presence or absence of rhA-IV (800 µg/ml) for 1 h. They were then incubated for a further 24 h with or without TBH (100 µM) and assessed for apoptotic response. Values are means ± SE from 4 separate experiments. abcP < 0.01, letters denote significant differences.

 
G6PD expression is required for Apo A-IV's antiapoptotic effects. Transfection of cells with an antisense phosphorothioated oligodeoxynucleotide to G6PD decreased expression of this enzyme to <23% of control levels, whereas transfection with a scrambled control construct did not significantly reduce G6PD expression (95.6% of control levels) (Fig. 8A). Transfection with the antisense construct significantly decreased both basal and Apo A-IV-stimulated G6PD activity by 73% and 80%, respectively (Fig. 8B). In both nontransfected cells and cells transfected with the scrambled control oligodeoxynucleotide, pretreatment with rhA-IV significantly attenuated TBH-induced apoptosis. Treatment with the antisense construct significantly increased basal (i.e., in the absence of TBH) levels of apoptosis (Fig. 9), and this increase was not affected by Apo A-IV (data not shown). However, rhA-IV's attenuation of TBH-induced apoptosis was completely reversed in cells treated with G6PD antisense (Fig. 9).



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  		Fig. 8. Graphs showing the effect of transfection of cells with G6PD antisense oligonucleotide on expression and activity of G6PD. G6PD-As denotes antisense phosphorothiolated oligodeoxynucleotide to G6PD; G6PD-Sc denotes control oligonucleotide (i.e., same base composition as A but in scrambled order). PC-12 cells were seeded into 2-cm2 cell culture wells (i.e., 24-well plate); 24 h later, at 50–70% confluence, they were transfected with 100 pM of either Sc or As constructs in OPTI-MEM medium (Invitrogen, Carlsbad, CA) using oligofectamine (Invitrogen) or treated with transfection reagent alone. After 48 h, cells were harvested for either Western blot analysis of G6PD expression (A, representative blot shown) or G6PD activity (B), with the latter being measured in the presence or absence of 800 µg/ml rhA-IV. Data in B are means ± SE from 3 separate transfections. abcP < 0.01, letters denote significant differences.

 


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  		Fig. 9. Effect of rhA-IV on TBH-induced apoptosis in cells expressing either antisense or control oligonucleotide in response to G6PD. PC-12 cells on glass coverslips in 24-well plates were transfected as described in the Fig. 8 legend; 48 h later they were treated with or without TBH (100 µM) in the presence or absence of rhA-IV (800 µg/ml). After 24-h treatment with TBH, cells were fixed and stained using DAPI as described in MATERIALS AND METHODS and then the percentage of apoptosis that had occurred was measured. Data are means ± SE from 4 separate transfection experiments at each condition. abcP < 0.05, letters denote significant differences.

 
Apo A-IV has no effect on cellular levels of G6PD protein. We used Western blot analysis to examine relative levels of G6PD protein in whole cell lysates from PC-12 cells treated with rhA-IV for graded periods up to 1 h, the same interval for which we observed an A-IV-induced increase in G6PD activity. Cellular levels of G6PD were unaffected by Apo A-IV (Fig. 10).



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  		Fig. 10. Effect of rhA-IV on G6PD protein expression. PC-12 cells were incubated with rhA-IV (800 µg/ml) for graded periods up to 60 min. They were then harvested and lysed, and equal amounts of lysate protein were subjected to SDS-PAGE followed by Western blot analysis. Blots were probed with goat anti-G6PD antiserum. A: representative blot from 3 separate experiments. B: densitometric measurement of G6PD Western blots. Values are means ± SE of 3 separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Although several recent studies have demonstrated protective effects of Apo A-IV in animal models of atherosclerosis (6, 9, 12, 21) and inflammatory bowel disease (32), the possibility that Apo A-IV might have direct cytoprotective effects had not been examined previously. Thus, in the present study, in which we used a simple, well-characterized model of cellular response to oxidant stress, we tested Apo A-IV's ability to protect.

We found that Apo A-IV could limit the cellular apoptotic response to two different oxidant chemicals: TBH, a model hydroperoxide, and diamide, a cell-permeant thiol oxidant. Similar protective effects against both of these compounds provided initial insight into Apo A-IV's likely mechanism of action. The oxidant TBH induces both mitochondrial ROS production and GSH redox imbalance (22–24), making it difficult to distinguish between an effect of A-IV on ROS compared with the redox state. However, at the concentration used in the present study, diamide is known to elicit GSH redox imbalance directly by oxidizing GSH to GSSG without any accompanying release of ROS (23). Thus A-IV's ability to attenuate TBH- and diamide-induced apoptosis similarly argues strongly in favor of an effect of Apo A-IV on intracellular GSH redox per se. Further support for this conclusion was provided by the finding that preincubation with Apo A-IV before oxidant challenge was protective, whereas addition of Apo A-IV to the cells 30 min after oxidant exposure was not. Pias and Aw (22, 23) showed that oxidants such as TBH and diamide produce a transient (i.e., within 30 min) but critical decrease in GSH redox (GSH-GSSG) that is restored by 60 min. This transient drop in GSH-GSSG is not only necessary for the eventual apoptotic response but also, once it has occurred, apoptosis is not reversible by antioxidant compounds. For example, if the imbalance is prevented by antioxidants such as NAC, apoptosis is prevented. However, if cells are treated with NAC after the transient imbalance has occurred and normal GSH-GSSG is restored, then apoptosis is not prevented (22). Clearly, prevention of oxidant-induced, transient GSH redox imbalance must occur within a narrow, strictly defined temporal window if eventual apoptosis is to be blocked. Thus, to the extent that Apo A-IV's effect depends on its being present before exposure to an oxidant, A-IV resembles NAC in its protective ability. Finally, direct measurements of GSH and GSSG confirmed that Apo A-IV can prevent oxidant-induced imbalance in GSH-GSSG. The transient rise in GSSG produced by TBH was completely abolished by Apo A-IV, such that the TBH-induced GSH-GSSG imbalance was prevented. Although Apo A-IV completely prevented the TBH-induced transient rise in GSSG, it had a negligible effect on absolute levels of intracellular GSH. This finding is likely due to the large absolute pool of intracellular GSH and is consistent with the inability of the GSH synthase inhibitor BSO to block Apo A-IV's antiapoptotic effect. Although we cannot yet rule out a possible effect of Apo A-IV on the relative distribution of intracellular GSH (e.g., cytoplasmic vs. mitochondrial) (10), our findings strongly suggest that Apo A-IV-mediated modulation of cellular GSH-GSSG is accomplished via reduction of GSSG. Altogether, the above results indicate that Apo A-IV inhibits oxidant-induced apoptosis in PC-12 cells by preventing imbalance in intracellular GSH redox.

The antiapoptotic effect of Apo A-IV was consistent, regardless of the source of Apo A-IV: rhA-IV, His-tagged A-IV, and native A-IV purified from serum were virtually identical in their effects. Thus the A-IV polypeptide is sufficient to exert protection. Moreover, the closely related apolipoprotein Apo A-I, which shares significant sequence similarity with Apo A-IV, was without effect, indicating a specific effect of Apo A-IV.

The effect of Apo A-IV was not reversed by washing Apo A-IV out of the media. Washout experiments were intentionally conducted using the lowest concentration of rhA-IV found to be antiapoptotic (200 µg/ml) to minimize the possibility that incomplete washing might have left residual Apo A-IV at levels sufficient to confer protection in the extracellular environment. We found that washout produced complete removal of Apo A-IV from the media. These results argue against direct scavenging or sequestering of oxidants by Apo A-IV before their entry into the cell and suggest a direct effect on cellular function that might be mediated by association of Apo A-IV with the cells.

The major enzyme directly involved in the reduction of GSSG to GSH is GSSG reductase, which catalyzes the reduction of 1 M GSSG to 2 M GSH, using reducing equivalents from NADPH. The supply of NAPDH in turn depends on the activity of several cellular dehydrogenase-6-phosphogluconate dehydrogenases, NADP+-dependent malate dehydrogenase, and G6PD, among which G6PD is quantitatively the most important (18). G6PD catalyzes the first, rate-limiting step of the pentose phosphate pathway, converting glucose-6-phosphate to 6-phosphogluconolactone and producing NADPH. The production of NADPH by the activity of G6PD is a critical regulator of intracellular GSH redox (18, 29, 30), and other studies have demonstrated that inhibition of G6PD directly by using inhibitors (23, 29) or by decreasing glucose availability (4, 23) elicits apoptosis. We measured the activities of GSSG reductase and G6PD in extracts from PC-12 cells incubated in the presence or absence of Apo A-IV. There was no effect of A-IV on the activity of GSSG reductase, but Apo A-IV elicited a 10-fold increase in G6PD activity, suggesting that Apo A-IV's effects on redox and apoptosis might be mediated by enhancing G6PD activity. This hypothesis was tested using both pharmacological and genetic approaches: inhibition of G6PD, either with DHEA or using G6PD antisense, completely abolished A-IV's effect. Altogether, these results support a novel action for Apo A-IV in the preservation of intracellular GSH redox balance via stimulation of G6PD activity.

The extent of stimulation of G6PD activity by Apo A-IV was remarkable and is more than sufficient to explain Apo A-IV's effects on GSSG levels and redox. The results of Western blot analysis indicated that induction of the G6PD protein is an unlikely explanation of Apo A-IV's stimulatory effect on G6PD activity and suggest a posttranslational mechanism (28, 31, 35). Currently, it is unknown how Apo A-IV might affect G6PD activity. Previous work suggests at least two possibilities: 1) modulation of reversible phosphorylation of G6PD similar to findings reported in response to high glucose (35) or 2) a shift in G6PD's intracellular distribution from a bound, inactive form to a soluble, active form as in the response to the fertilization in oocytes (28) or to growth factors in endothelial cells (31). The results of our washout experiments suggest that regardless of the mechanism of Apo A-IV's effect on G6PD, it may involve cellular uptake of Apo A-IV protein. Work to determine the mechanism underlying the novel stimulatory effect of Apo A-IV on G6PD is ongoing in our laboratory.

Because oxidant-induced apoptosis could be considered a self-sacrificing protective mechanism to limit or to prevent a significant inflammatory response secondary to cell necrosis, Apo A-IV's antiapoptotic effects might appear to argue against an anti-inflammatory role for this protein. However, with regard to cellular responses to oxidants, mechanisms leading to apoptosis vs. inflammation and/or necrosis likely diverge from a common set of upstream events. Whether such events lead to eventual apoptosis or inflammatory activation may depend on the cell type, the state of differentiation and/or mitotic competence, the prevailing physiological state, or the extent and/or duration of oxidant stress (2, 5). Regulation of GSH redox is a logical candidate for modulation of such an upstream event because it is one of the first lines of defense against oxidant challenge. We propose that Apo A-IV can modulate this early event in the overall cellular response to oxidants. This hypothesis is consistent with the observed rapid kinetics (i.e., within 5 min) of oxidant-induced GSH redox imbalance and the ability of Apo A-IV to prevent this imbalance and attenuate the downstream biological end point (i.e., apoptosis). If Apo A-IV's actions in the undifferentiated PC-12 model reflect a fundamental redox-modulatory role for this protein, then its antiapoptotic activity does not rule out an anti-inflammatory role for this protein mediated through a similar mechanism, i.e., preservation of intracellular GSH redox balance. Further work is necessary to test this hypothesis.

In summary, we have shown a significant and specific effect of apolipoprotein A-IV in modulating oxidant-induced apoptosis in undifferentiated PC-12 cells, which can be explained by A-IV-mediated preservation of intercellular GSH redox balance via stimulation of G6PD activity. In our ongoing studies, we are examining whether this novel effect of Apo A-IV reflects a general redox-modulatory role for this protein that could underlie some if not all of its previously reported protective effects.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R29 DK-52148 (to T. J. Kalogeris) and R01 DK-44510 (to T. Y. Aw) and by funds from the Louisiana State University Health Sciences Center Department of Surgery.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Fu Qin Duan, William Goodwill, Dr. Masahiro Okouchi, and Cynthia Rodriguez for technical assistance. We also thank Nicholas Duverger for generously providing the pL2102-rhA-IV plasmid.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. J. Kalogeris, Dept. of Surgery, Louisiana State Univ. Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130 (e-mail: tkalog{at}lsuhsc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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