Short-term exposure of 4-hydroxynonenal induces mitochondria-mediated apoptosis in PC12 cells

Abstract

4-Hydroxynonenal (4-HNE) is one of the most reactive aldehydic by-products of lipid peroxidation. The role of 4-HNE in the etiology of various neurodegenerative disorders including cerebral ischemia/reperfusion, Alzheimer’s disease, Parkinson’s disease, etc. has been documented. We and others have reported that long-term toxic insults of 4-HNE triggers apoptotic signals and oxidative stress in various cells. However, the status of apoptosis following short-term exposure and underlying mechanisms has not been explored so far. We studied the apoptotic changes in PC12 cells receiving short-term exposure of 4-HNE. A significant dose-dependent induction in reactive oxygen species (ROS) and early response markers (c-Fos, c-Jun, and GAP-43) were observed in cells exposed to 4-HNE (10, 25, and 50 µM) for 1h. Following the exposure of PC12 cells to 4-HNE, the levels of protein and messenger RNA expressions of P⁵³, Bax, and caspase 3 were significantly upregulated, whereas the levels of Bcl₂ was downregulated. We could record the apoptotic signals and ROS generation in PC12 cells receiving 4-HNE exposure for such a short period of time. Induction in the expression and activity of caspase 3 has also indicated the mitochondrial mediation in the apoptosis induction.

Introduction

Aldehydic products of lipid peroxidation of biological membranes have been reported as important etiological factors in numerous neurodegenerative disorders.^1–3 Of these, 4-hydroxynonenal (4-HNE), a long-chain α, β-unsaturated aldehyde, is known to be most toxic. ^4,5 Higher concentrations of 4-HNE are cytotoxic and causes oxidative stress-mediated cell death in a variety of cell types including PC12 cells. ^6–9 The low-level exposure of 4-HNE also modulates intracellular signaling by activating the mitogen activated protein kinases (MAPK), stress-activated protein kinase and c-Jun N-terminal protein kinase cascades, and inhibiting the nuclear factor κ-B activity.^7,10,11 We and others have shown the association of 4-HNE-induced oxidative stress-mediated cytotoxicity/genotoxicity with dopamine (DA-D₂), cholinergic (muscarinic), benzodiazepine, and serotonin (5-HT)-2A receptors in cultured cells of human and animal origins.^8,12 The expression of GSTP1-1 and increased levels of intracellular Ca⁺⁺ are also associated with 4-HNE exposure in cells. ⁹

In our earlier studies, a concentration-dependent metabolism of 4-HNE in PC12 cells was observed in the form of glutathionyl conjugates and 4-Hydroxy-trans-2-nonenoic acid (HNA) and 1,4-Dihydroxy-2-nonene (DHN) metabolites within 1 h of exposure. ¹³ Although, 4-HNE has been reported to induce apoptosis ^{14 –16} and necrosis,^17–19 the underlying mechanisms following short-term exposure of 4-HNE is not known. Thus, the present study was carried out to test whether the expression of selected apoptosis markers in PC12 cells, receiving short-term exposure of 4-HNE is altered. Our results show significant dose-dependent alterations in reactive oxygen species (ROS), and early responses to specific apoptotic markers in cells exposed to 4-HNE. Furthermore, enhanced expression and activity of caspase 3 following 4-HNE exposure to cells indicates the mitochondrial mediation in the apoptosis induction. Together, the results from these studies may provide a mechanism through which 4-HNE induces apoptosis in neuronal cells.

Materials and methods

Cell culture

PC12 cells used in the study were originally procured from National Centre for Cell Sciences, Pune, India, and grown in Nutrient Mixture/F-12 (Hams) supplemented with 2.5% fetal bovine serum (FBS), 15% horse serum, 0.2% sodium bicarbonate, and antibiotic/antimycotic solution (100×, 1 ml/100 ml of medium; Invitrogen, Life technologies, Staley Road, Grand Island, NY 14072, USA). The cells were maintained in 5% CO₂–95% atmosphere under high humidity at 37°C. Cells were assessed for cell viability by trypan blue dye exclusion assay as described earlier by Pant et al. ²⁰ and batches showing viability of more than 95% were used in the experiments.

Reagents and consumables

All the specified chemicals, reagents, and diagnostic kits were procured from Sigma Chemical Company Pvt. Ltd. (St. Louis, Missouri, USA), unless otherwise stated. Nutrient mixture F-12 Hams, antibiotics/antimycotics solution (100×), FBS (Staley Road, Grand Island, NY, USA), and horse serum were purchased from Gibco BRL (Kamstrupvej 90, Denmark-4000, Roskilde, USA). Culture wares and other plastic consumables used in the study were procured from Nunc (Denmark). Milli-Q water was used in all the experiments. 4-HNE, an unsaturated aldehyde, was generously gifted by Dr Sanjay Srivastava, Department of Cardiology, University of Louisville (Kentucky, USA).

Experimental design

PC12 cells were exposed to various concentrations (1–50 μM) of 4-HNE for 1 h. Cells receiving 4-HNE insult were then analyzed for ROS generation and to determine the changes in the expression of early response proteins (c-Fos, c-Jun, and GAP-43) as well as selected marker genes associated with mitochondria-mediated apoptosis using real time-polymerase chain reaction (RT-PCR). The possible associations of these proteins in 4-HNE-induced apoptosis were also assessed using immunoblot–Western blot analysis.

ROS generation

ROS generation was assessed using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma Aldrich (St. Louis, Missouri, USA), USA) dye as a fluorescence agent following the protocol earlier described by us. ²¹ In brief, cells (5 × 10 ⁴ per well) were allowed to adhere to poly-l-lysine-coated eight-well chamber slide flasks. Following the exposure of 4-HNE for 1 h, cells were washed with phosphate-buffered saline (PBS) and incubated for the next 30 min in DCFH-DA (20 μM) containing incomplete culture medium in the dark. Then, the slides were analyzed for intracellular fluorescence using an upright fluorescence microscope (Nikon Eclipse 80i (Yokohama, Japan) equipped with Nikon DS-Ri1 12.7 megapixel camera). Image analysis software Leica Qwin 500 (Leica Microsystem, Wetzlar, Germany) was used to quantify the change in the fluorescence and data were expressed as percentage change in the fluorescence area.

Transcriptional changes

Alterations in the messenger RNA (mRNA) expression of apoptosis marker genes (P⁵³, Bax, Bcl₂, and caspase 3) were studied following the protocol as described earlier by us.^21,22 In brief, cells (1 × 10 ⁶ ) were allowed to grow in six-well culture plates unless reached to 80–85% confluence. Following the exposure of cells to 4-HNE for 1 h, total RNA was isolated from both experimental and control sets using GeneElute mammalian total RNA Miniprep Kit (catalog no. RTN-70, Sigma). The purity and yield of RNA was assessed by Nanodrop ND-1000 Spectrophotometer V3.3 (Nanodrop Technologies Inc., Wilmington, Delaware, USA). RNA quality in terms of integrity was also checked by running RNA onto 2% denaturing agarose gel. Total RNA (2 µg) was reverse-transcribed into cDNA by SuperScript III first-strand cDNA synthesis Kit (catalog no. 18080-051, Invitrogen Life Science, USA). qRT-PCR was performed by SYBR Green dye (ABI, Applied Biosystem, 850 Lincoln Centre Drive Foster City California, USA) using ABI PRISM^® 7900HT Sequence Detection System (Applied Biosystems, USA). Real-time reactions were carried out in triplicate. Specificity of primer sets and genomic DNA contamination were assessed for all the samples by analyzing the melting curve and no template control (NTCs), respectively. GAPDH was used as internal control to normalize the data. 4-HNE-induced alterations in mRNA expression were expressed in terms of relative quantity.

Protein expression (Western blotting)

Alterations in the expression of selected marker proteins (P⁵³, Bax, Bcl₂, and activated caspase 3) associated with apoptosis were carried out by Western immunoblotting as described earlier. ¹⁶ In brief, after respective exposure, cells were washed twice with ice-cold PBS (pH 7.4) and centrifuged at 1000 rpm for 10 min. Cell pellets were lysed using cell lysis reagent (catalog no. C2978, Sigma) in the presence of 1× protein inhibitor cocktail (catalog no. P8340; Sigma). An equal amount (50 μg/well) of proteins was loaded in 10% Tricine–sodium dodecyl sulfate gel and electrophoresis was carried out. Following electrophoresis, protein bands from the gel were transferred on polyvinylidene fluoride membrane (Millipore, Bedford, Massachusetts, USA) in an electrophoresis transfer apparatus (BioRad, Hercules, California, USA). The membrane was blocked overnight in Tris-Buffered Saline Tween-20 (TBST) (30 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) containing 5% nonfat milk. Membranes were then probed with primary antibodies specific for Bax (1:500, Santa Cruz, USA), Bcl₂, activated caspase 3 (1:1000, Cell Signaling Technology (CST), USA), and β-actin (1:2000, Santa Cruz). After several washings with TBST, membrane was incubated with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit for Bax and cleaved-caspase 3, goat anti-mouse for all others) for 2 h at room temperature. Then, the blots were developed using 3,3′,5,5′-tetramethylbenzidine (TMB)–hydrogen peroxide (H₂O₂; Sigma Weltevreden Park, Johannesburg, 1715, S.A.). Pictures of specific band were taken by gel documentation system (Alpha Innotech, USA) and densitometric analysis was done by AlphaEase™ FC StandAlone V. 4.0.0 software.

Protein expression (Immunocytochemistry)

Immunocytochemical localization for early response gene proteins (c-Fos, c-Jun, and GAP-43) was carried out using specific antibodies following the protocol. ²² In brief, 4-HNE exposed cells were fixed in 4% paraformaldehyde for 10 min. Cells were washed with PBS thrice and incubated with 0.5% H₂O₂ (w/v) in methanol to quench endogenous peroxidase. Nonspecific binding sites were blocked by incubating cells with 0.5% bovine serum albumin and 0.1% Triton X-100 in phosphate buffer saline (PBS) for 2 h. Cells were then incubated in primary monoclonal antibodies specific for c-Fos, c-Jun, and Gap-43 proteins (1:200, Sigma Chemicals Company Pvt. Ltd.) for 1 h. Following the washing with PBS, cells were again reincubated with goat anti-rabbit horseradish peroxidase conjugate secondary antibody (1:500) for 2 h. Cells were washed with PBS to remove any unbound secondary antibody and incubated with diaminobenzidine hydrochloride for 5–15 min to develop the color. Cells were visualized and images were captured using an upright microscope (Nikon Eclipse 80i equipped with Nikon DS-Ri1 12.7 megapixel camera). Image analysis software Leica Qwin 500 was used to quantify the percentage change in the area of expression of protein early response proteins. Similar experiments in cells without any exposure were run under parallel and served as controls.

Caspase 3 activity

4-HNE-induced alterations in the activity of caspase 3 were monitored using kits (Biovision, catalog no. K106, USA). Following 4-HNE exposure for 1 h, cells were pelleted, resuspended in prechilled extraction buffer (50 μl), and incubated for 10 min on ice. Then, the samples were centrifuged for 5 min at 500g and the clear supernatant (50 μl per well) transferred to 96-well culture plates. Assay buffer (50 μl) and substrate conjugate (5 μl) were added and mixed well. Immediately after the completion of 2 h incubation at 37°C in dark, the contents were mixed thoroughly and read for absorbance at 400 nm. The values of exposed groups were compared with unexposed control sets and the data expressed as a percentage of control.

Statistical analysis

Results are expressed as mean ± standard error (SE) of the values obtained from three independent experiments, and in each experiment, a minimum of four replicates were used. Statistical analysis was performed using one-way analysis of variance, and post hoc Dunnett’s test was applied to compare values between control and treated groups. The values of p < 0.05 were considered as statistically significant.

Results

ROS generation

Statistically significant (p < 0.001) ROS generation was observed in PC12 cells receiving 4-HNE (10, 25, and 50 μM) exposure for 1 h (Figure 1(a) and (b)). The increase in ROS generation was dose dependent, i.e. 127.0 ± 8.6%, 146.0 ± 9.8%, and 178.0 ± 12.1% of unexposed control sets following the exposure of 10, 25, and 50 μM of 4-HNE, respectively.

Figure 1.

(a) 4-HNE-induced ROS generation in PC12 cells. Cells were exposed to various concentrations of 4-HNE for 1 h. ROS generation was studied using dichlorofluorescin diacetate dye. Images were taken using Nikon fluorescence microscope (model 80i) attached with 12.7 Megapixel Nikon DS-Ri1 digital CCD cool camera. (I): unexposed cells (control); (II): cells exposed to 4-HNE (1 μM); (III): cells exposed to 4-HNE (10 μM); (IV) cells exposed to 4-HNE (25 μM); and (V) cells exposed to 4-HNE (50 μM). (b) Relative quantification of ROS generation in PC12 cells following various concentrations of 4-HNE for 1 h. Quantification of fluorescence images of intracellular ROS was done using Leica Q Win500 image analysis software. *p < 0.01, **p < 0.001 versus control. 4-HNE: 4-hydroxynonenal; ROS: reactive oxygen species.

Transcriptional changes in apoptosis markers

The alterations in the levels of mRNA expression of selected apoptosis marker genes in PC12 cells exposed to 4-HNE for 1 h are presented in Figure 2(a) to (d). Significant (p < 0.001) upregulation in the expression of proapoptotic genes, i.e. caspase 3 (3.55 ± 0.15-fold), Bax (3.12 ± 0.25-fold), and p53 (2.96 ± 0.21-fold), was observed at 50 μM concentration, whereas antiapoptotic gene Bcl2 (0.25 ± 0.01-fold) was significantly downregulated at this dose. In general, 4-HNE-induced alterations were dose dependent at 10, 25, and 50 μM concentrations, while the lowest concentration used, i.e. 1 μM was found to be ineffective.

Figure 2.

RT-PCR analysis to study the alterations in the expression of mRNA of various genes, namely, P53 (a), Bax (b), Bcl-2 (c), and caspase 3 (d) in PC12 cells exposed to different concentrations of 4-HNE for 1 h. The data provided are mean ± SE from three separate experiments. *p < 0.05, **p < 0.001. RT-PCR: real-time polymerase chain reaction; 4-HNE: 4-hydroxynonenal.

Translational changes in apoptosis markers

To further determine whether these changes in the apoptotic marker genes are happening at the level of protein expression, we carried out immunoblot analyses for these proteins. The results of our immunoblot analyses are shown in Figure 3(a) to (d). Cells exposed to 4-HNE (10, 25, and 50 μM) for 1 h showed a significant upregulation in the expressions of Bax (1.5 ± 0.15, 2.6 ± 0.19, and 2.4 ± 0.2-fold), caspase 3 (1.4 ± 0.13, 2.1 ± 0.2, and 3 ± 0.26-fold), and p53 (1.8 ± 0.13, 1.7 ± 0.14, and 2.1 ± 0.16-fold), whereas the expression of Bcl₂ protein was downregulated by 0.8 ± 0.06, 0.4 ± 0.03, and 0.2 ± 0.019-fold, respectively. Similar to transcriptional changes, 4-HNE-induced alterations at the protein level were dose dependent and exposure of 1 μM for 1 h was found to be ineffective.

Figure 3.

4-HNE-induced alterations in the protein expression of apoptosis markers, namely, P53 (a), Bax (b), Bcl-2 (c), and activated caspase 3 (d) in PC12 cells. Cells were exposed to various concentrations of 4-HNE for 1 h. Cells were harvested and subjected to Western blotting using respective antibodies. Relative optical density was determined by densitometric analysis. Quantification was done with a gel documentation system (Alpha Innotech) with the help of AlphaEase FC StandAlone V. 4.0 software. Data are expressed as mean ± SE. *p < 0.05, **p < 0.01. 4-HNE: 4-hydroxynonenal.

Translational changes in early response markers

Cells exposed to 4-HNE (10, 25, and 50 μM) for 1 h showed significant upregulation in the expressions of c-Fos (8.87- ± 0.62-, 19.67- ± 1.2-, and 27.94- ± 1.9-fold), c-Jun (8.67- ± 0.64-, 19.52- ± 1.2-, and 27.27- ± 1.7-fold), and GAP-43 (16.57- ± 1.2-, 26.27- ± 1.9-, and 30.08- ± 2.1-fold), respectively. However, there were no alterations in the expression of these gene proteins in cells expose to 4-HNE at 1 μM concentration for 1 h (Figure 4(a) and (b)).

Figure 4.

(a) Representative microphotographs of immunocytochemical localization of early response gene proteins in PC12 cells following the exposure of 4-HNE (1–50 μM) for 1 h. (b) Expression of early response genes in PC12 cells following 4-HNE (1–50 mM) exposure for 1 h. All values represent the mean ± SE obtained from the images of at least 20 microscopic fields and analyzed by Leica Q-Win 500 image analysis software. *p < 0.05, **p < 0.001. 4-HNE: 4-hydroxynonenal.

Activity of caspase 3

Highlights of 4-HNE-induced alterations in the activity of caspase 3 are shown in Figure 5. The activity was found to be increased with increasing doses of 4-HNE. The affects were statistically significant for 4-HNE exposures at 25 and 50 μM (1.77- ± 0.15- and 2.39- ± 0.23-fold of unexposed controls, respectively).

Figure 5.

Induction in the activity of caspase 3 in PC12 cells exposed to 4-HNE (1–50 µM) for 1 h. Cells exposed to camptothecin (1 µg/mL) for 24 h were used as positive control. *p < 0.05; **p < 0.001. 4-HNE: 4-hydroxynonenal.

Discussion

4-HNE-induced apoptosis and oxidative stress are documented in neuronal cells.^7,23 We have also reported earlier that 4-HNE at higher concentrations induce cytotoxicity in PC12 cells, which was found to be associated with the altered levels of neurotransmitter receptors such as dopamine, cholinergic, serotonin, and benzodiazepine receptors. ⁸ In addition, we have reported the oxidative stress-mediated cytotoxic responses in PC12 cells exposed to various doses of 4-HNE. ⁹ In our earlier studies, a concentration-dependent metabolism of 4-HNE in PC12 cells has also been demonstrated within 1 h of exposure in the form of glutathionyl conjugates and HNA and DHN metabolites. ¹³ In this study, we are reporting the expressional changes in apoptosis marker genes in PC12 cells exposed to various concentrations of 4-HNE for such a short period of 1 h only.

ROS generation is considered to be one of the key signals for oxidative stress-induced apoptosis. ²⁴ In the present investigation, significant dose-dependent generation of ROS was observed in PC12 cells exposed to 4-HNE for 1 h. Our results confirm the findings of Feng et al. ²⁵ and Uchida et al. ²⁶ who have also reported ROS generation-mediated oxidative stress in cells exposed to higher concentrations (25 and 50 µM) of 4-HNE. However, in their experiments, the exposure periods were more than 1 h. Similar to our findings, they have also shown that lower doses of 4-HNE, i.e. <1 μM are ineffective. Induced levels of ROS are well-known etiological factors associated with oxidative stress and are known to cause cell death via apoptosis in a variety of cell types.^21,27,28 Thus, ROS generation in the present study may be a mediator for 4-HNE-induced apoptosis in neuronal cells.

The involvement of mitochondrial chain complexes in ROS-induced apoptotic changes in cytoplasm has been reported. ²⁹ Such apoptotic changes are known to follow different pathways.^24,30 The mitochondrial-dependent apoptotic pathway is also known to be involved in 4-HNE-induced cytotoxicity in various cells^31,32 including PC12 cells.^16,23 We observed that 4-HNE significantly upregulates the expression (mRNA and protein) of P⁵³, Bax, caspase 3, and downregulated Bcl₂ in a dose-dependent manner. In general, transcriptional changes were well coordinated with translational changes and with physiological activity of caspase 3. The upregulation of nuclear P⁵³ protein is known to play an important role in minimizing DNA damage by inducing transcriptional reprogramming, which eventually leads to controlled cell death.^33–35 However, higher level of cytoplasmic P⁵³ protein interacts with mitochondria, thereby promoting mitochondrial membrane permeabilization ³⁶ and plays an important role in the regulation of apoptosis. ³⁷ This cytoplasmic P⁵³ protein has been suggested to induce proapoptotic members of the Bcl₂ family such as Bax and Bak, and their displacement with antiapoptotic Bcl₂ proteins. ³⁸ Thus, the alterations in the expression profile of marker genes in this study indicates that P⁵³ triggers the mitochondrial apoptotic cascade in PC12 cells exposed to 4-HNE. Similar to our findings, 4-HNE-induced apoptosis in other cell types has also been suggested through the P⁵³-dependent intrinsic pathway.^11,39 The induction of P⁵³ by 4-HNE followed by the activation of caspase 3 and the onset of apoptosis has also been reported by Li et al. ⁴⁰ Association of 4-HNE-induced apoptosis with P⁵³ accumulation suggests that the process of apoptosis elicited by 4-HNE may be due to the changes in the availability or sensitivity of specific regulatory pathways that are activated by the 4-HNE.

Initially, P⁵³ is activated in response to DNA damage by 4-HNE, and these upregulated levels of P⁵³ promote induction in the expression of proapoptotic Bax, while downregulating the expression of antiapoptotic Bcl₂ protein. This imbalance in the ratio of Bax/Bcl-2 could lead to the dissipation in mitochondrial membrane potential. Finally, dysfunctional mitochondria could release cytochrome-c in cytosol, which could activate caspase 3 via the activation of procaspase 9. In the case of 4-HNE-induced apoptosis, an imbalance in Bax/Bcl-2 protein may be an upstream event followed by mitochondrial-mediated activation of caspase 3, a final executer caspase, which leads to cell apoptosis. Activation of P⁵³ is associated with a rapid increase in its levels and with an increased ability to bind DNA and mediate transcriptional activation of apoptotic signals.^21,41

Induction in the levels of early response gene proteins such as c-Fos, c-Jun, and GAP-43, which are integral components of transcription factor AP-1, has been discussed as a regulator of cell death, survival, and regeneration. ⁴² In our present study, exposure of 4-HNE-induced expression of c-Fos, c-Jun, and GAP-43 shows the effects of 4-HNE as a toxicant and could be well correlated with previously reported induced expression of these genes in the appearance of neurotoxicity.^22,43 Oxidation products of 4-HNE thereby may have a role in regulating cell death/survival as in the case of neurodegeneration.

Together, our data provide insights to the induction of apoptotic damages in neuronal cells following short-term exposure (1 h) of 4-HNE, which may help understand the possible cellular and molecular mechanisms involved in 4-HNE-induced neuronal damage.