Bid Mediates Anti-Apoptotic COX-2 Induction Through the IKKβ/NFκB Pathway Due to 5-MCDE Exposure

Abstract

Although Bid is considered to be a cell apoptotic mediator, current studies suggest that it has a possible role in cell survival for mouse embryonic fibroblasts (MEFs) in response to low doses of anti-(±)-5- methylchrysene-l,2-diol-3,4-epoxide (<0.25µM) (5-MCDE). We found that the exposure of MEFs to 0.25 µM 5-MCDE resulted in a slight apoptotic induction, while this apoptotic response was substantially increased in the Bid knockout MEFs (Bid^−/−), suggesting that there is a Bid-mediated anti-apoptotic function in this response. This notion was further supported by the findings that re-constitution expression of Bid into Bid^−/− cells could inhibit the increased apoptosis. Further studies show that the antiapoptotic function of Bid was associated with its mediation of COX-2 expression. This conclusion was based the reduction of COX-2 expression in Bid−/− cells, the restoration of low sensitivity to 5-MCDE-induced apoptosis by the introduction of Bid into Bid−/− cells, and increased sensitivity of WT MEFs to 5-MCDE-induced apoptosis by the knockdown of COX-2 expression. Furthermore, we found that Bid mediated COX-2 expression through the IKKβ/NFκB pathway because the deficiency of Bid in Bid−/− MEFs resulted in the blockade of IKK/NFκB activation and knockout of IKKβ caused abrogation of COX-2 expression induced by 5-MCDE. Collectively, our results demonstrate that Bid is critical for COX-2 induction through the IKKβ/NFκB pathway, which mediates its anti-apoptotic function, in cell response to low doses of 5-MCDE exposure.

INTRODUCTION

Apoptosis plays an important role in many physiologic and pathologic processes [1, 2]. It is accepted that alterations of the balance between cell survival and apoptosis contribute to the pathogenesis of a number of human diseases that include autoimmune diseases, neurodegenerative disorders, and acquired immunodeficiency syndrome, as well as cancers [2]. Apoptosis is implicated in anti-cancer activity because it can eliminate cells that are genetically damaged because of exposure to carcinogens, thereby preventing their replication and the accumulation of abnormal cell clones.

The decision of a cell to undergo apoptosis is made by the cross-talk between cell survival signaling and death signaling, a process which could be influenced by a wide variety of signaling pathways. It is generally assumed that the Bcl-2 family proteins play an important role in the regulation of cellular apoptosis and subsequently that they are implicated in carcinogenesis [3]. Bid belongs to the “BH3-only” proapoptotic Bcl-2 family. Previous studies have shown that Bid is not only able to mediate cell apoptosis, it may also be involved in the regulation of cell proliferation and genomic stability [4, 5]. Bid is a key component for mitochondrial apoptotic pathways in cell response to intracellular stress such as carcinogens. In the process of Bid-mediated cell apoptosis, caspase-8 can cleave Bid into a truncated protein (tBid), which subsequently translocates to the mitochondria and causes cell apoptosis [6]. Our current studies provide a novel scenario of the anti-apoptotic function of Bid through the activation of the IKKβ/NFκB pathway, which further mediates COX-2 expression, in cellular response to relatively low dose (0.25µM) of anti-(±)-5- methylchrysene-1,2-diol-3,4- epoxide (5-MCDE) in mouse embryonic fibroblasts (MEFs).

MATERIALS AND METHODS

Cell Culture

Immortalized wild type (WT) and Bid deficient (Bid^−/−) mouse embryonic fibroblasts (MEFs) were generated as described in previous studies [7], the IκB kinaseβ (IKKβ) deficient (IKKβ^−/− MEFs) were a gift from M. Karin (University of California, San Diego, La Jo11a, CA [8]. These MEFs, as well as their stable transfectants, were maintained at 37°C in 5% CO₂ in an incubator with Dulbecco's modified Eagle’s medium (DMEM, Calbiochem, San Diego, CA), supplemented with 10% fetal bovine serum (FBS, Nova-Tech, Grand Island, NE), 1% penicillin/streptomycin, and 2 mML-glutamine (Life Technologies, Inc. Rockville, MD).

Reagents and Antibody

(±)-anti-5-methylchrysene-l,2-diol-3,4-epoxide (5-MCDE) was synthesized as described in our previous studies [1, 9], and dissolved in DMSO to get a stock concentration at 0.5 mM [8, 10]. Luciferase assay substrate was purchased from Promega (Madison, WI). The antibodies against phospho-IKKα/β, IKKα, IKKβ, phosphor-JNKs, JNKs, phosphor-c-Jun, c-Jun, phospho-p38 kinase, p38 kinase, caspase-3, PARP, GFP, and Bcl-2 were purchased from Cell Signaling Technology (Beverly, MA). The antibody against Bid was purchased from BD Biosciences. Anti-HA was purchased from Upstate Biotechnology, Inc (Lake Placid, NY) and anti-P-Actin antibody was obtained from Sigma (St Louis, MO). Anti-COX-2 antibody was purchased from Cayman Chemical (Ann Arbor, MI).

Plasmids and Stable Transfection

The plasmid expressing the full length of IKKβ (HA-IKKP) was described in a previous study, and its stable transfectant was established and identified as described in our published studies [10]. The plasmid expressing the full-length Bid (pEGFP-C3-Bid) was a gift from Dr. Andrew Gilmore (University of Manchester, United Kingdom) [11]. The nuclear factor-kappa B (NFκB)-luciferase reporter plasmid was constructed as previously described [11, 12]. The specific small interference RNAs targeting COX-2 were described in our previous study [12]. Cell transfection was performed with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instruction. Briefly, MEFs were cultured in a six-well plate to 80% to 90% confluence. Five micrograms of plasmid DNA in combination with hygromycin resistant LacZ plasmid were mixed with 10 µL of Lipofectamine 2000 reagent and used to cotransfect each well in the absence of serum. After 6 hours, the medium was replaced with 10% FBS DMEM. Approximately 36 hours after the beginning of the transfection, cultures were subjected to hygromycin (100µg/ml) selection, and hygromycin resistant cells from the selection were pooled as a stable mass. These stable transfectants were cultured in the hygromycin free medium for at least two passages before used for following experiments.

Luciferase Reporter Assay

The confluent monolayer of MEF NFκB Luc mass1 was trypsinized, and 8×10³ viable cells suspended in 100 µl DMEM supplemented with 10% FBS were added to each well of 96-well plates. After the cell density reached 80% to 90%, the cell culture medium was replaced with 0.1% FBS DMEM. Twelve hours later, cells were exposed to 5-MCDE at a final concentration, as indicated in the figures for NFκB induction. At different points after treatment, cells were lysed and the luciferase activity was measured using Promega luciferase assay reagent with a luminometer (Wallac 1420 Victor2 multipliable counter system). The results are expressed as NFκB activity relative to medium control (relative NFκB activity) [1].

Reverse Transcription Polymerase Chain Reaction (RT-PCR)

MEFs suspended in 10% FBS DMEM were added to each well of 6-well plates. After the cell density reached 80%-90%, the cells were treated with 5-MCDE at a final concentration as indicated in 0.1% FBS DMEM. Twelve hours post exposure to 5-MCDE, cells were collected and total RNA was extracted from the cells using Trizol reagent (Invitrogen, Carlsbad, California). cDNA were synthesized by the ThermoScript™ RT-PCR system according to manufacturer’s instructions (Invitrogen). A pair of oligonucleotides (5’-tcc tcc tgg aac atg gac tc −3’ and 5’-gct cgg ctt cca gta ttg ag −3’) were used as the specific primers to amplify mouse COX-2 cDNA. The mouse β-actin cDNA fragments were amplified by the primers 5’-gacgatgatattgccgcact-3’ and 5’-gataccacgcttgctctgag-3’.

Electrophoretic Mobility Shift Assays (EMSA)

MEFs suspended in DMEM supplemented with 10% FBS were added to 10-cm dishes. After the cell density reached 80% to 90%, the cells were treated with 5-MCDE in 0.1 % FBS DMEM for the time indicated in the figure legends. Nuclear proteins were prepared with the Cellytic™ Nu-CLEAR™ Extraction Kit (Sigma, Saint Louis, Missouri, USA) following the manufacturer’s protocols. Briefly, MEFs were washed in PBS and the pellets of cells were suspended in 1×Lysis buffer A (10 mM HEPES (pH 7.9) with 1.5 mM MgCl₂, 10 mM KCl, 0.1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and PMSF, then incubated for 20 min on ice. After that, 10% Nonidet P-40 (final concentration of 0.6%) was added. The suspension was vortexed vigorously for 10 seconds and centrifuged immediately for 30 seconds at 11,000×g. The crude nuclei pellet was suspended in Extraction Buffer (20 mM HEPES, pH 7.9 with 1.5 mM MgCl₂, 0.42 M NaCl, 0.2 mM EDTA, 25% (v/v) Glycerol, 0.1 M DTT, and 0.1 mM PMSF) and centrifuged. The probes used for EMSA were radiolabeled by [γ−³²P] ATP with T4 polynucleotide kinase. The synthetic oligonucleotides used as probes for NFκB binding in EMSA experiments are 5’-ctccccaacccgggcgttccccagcgagg-3’. For EMSA assay, 10 µg of nuclear protein was subjected to the gel shift assay by incubating with 1 µg of poly(dI-dC) DNA carrier in DNA-binding buffer (10 mM Tris, pH 8.0, 150mM KCl, 2mM EDTA, 10mM MgCl₂,10 mM DTT, 0.1% BSA, and 20% glycerol) in a final volume of 10 µl on ice for 10 mins. Then 10⁵ cpm of the ³²P-labeled double-stranded oligonucleotide (2 µ1) were added and the reaction system was incubated at room temperature for 30 min. For competition experiments, 20-fold molar excess of the unlabeled oligonucleotides or the irrelevant cold probes were added before the addition of the probe. For the super-gel shift assay, nuclear extracts were incubated with 2µg of either the pre-immune serum or the indicated antibodies for 30 min at 4°C before the addition of the probe. The anti-p65 antibodies specific for the super-gel shit assay were developed by Santa Cruz Biotechnology (Santa Cruz, CA). DNA-protein complexes were resolved by electrophoresis in 5% nondenaturing glycerol-polyacrylamide gels.

Western Blot

MEFs (3 × 10⁵) were cultured in each well of 6-well plates to 80–90% confluence. The cell culture medium was replaced with 0.1% FBS DMEM and incubated for 12h. The cells were exposed to 5-MCDE (0~0.5 µM) for time indicated in the figure legends, and thereafter were washed once with ice-cold PBS and extracted with the sample buffer. The cell extracts were separated on polyacrylamide-SDS gels, transferred and probed with primary antibodies as indicated. The protein bands specifically bound to primary antibodies were detected by using an anti-rabbit IgG (AP-linked) and by an ECL Western blotting system (Amersham Biosciences, Piscataway, NJ) [12].

Cell Death Analysis

To analyze the apoptotic cells by using propidium iodide (PI) staining, MEFs were seeded into each well of 6-well plates and cultured in 10% FBS MDEM until 80–90% confluence. After exposure to 5-MCDE for the indicated time, the cells were collected and fixed in ice-cold 75% ethanol at −20°C overnight. The fixed cells were stained in the buffer containing 100 mM sodium citrate, 0.1% Triton X-100, 0.2 mg/ml RNAse A, and 50 µg/ml PI at 4 °C for 1 h and then analyzed by the Epics XL FACS (Beckman Coulter, Miami, FL) as described in our previous publications.

MTT Cell Viability Assay

A MTT cell viability assay kit (Biotium Inc, Hayward, USA) was used to detect the cell viability according to the manufacture’s protocol. Briefly, the MEFs (1×10⁴) were seeded into each well of 96-well plates. After 24 h of incubation, the cells were treated with either different doses of 5-MCDE compound or 0.1% dimethyl sulfoxide (DMSO) for 24 hrs. The medium was then replaced with 100 µl of fresh medium incubated with 10 µM MTT at 37 °C for 4 h. The medium was then removed, and 200 µl DMSO was added into each well. The optical absorbance was determined using ELISA plate reader (Tecan Infinite F200, Switzerland) at the wavelength of 570 nm. The absorbance directly correlates with the cell viability.

Statistical Analysis

The significances of the difference between different groups were determined with the ANOVA. The differences were considered significant at P<0.05.

RESULTS

Bid Mediated Anti-Apoptotic Response to Relative Low Doses of 5-MCDE Exposure in MEFs

Bid is initially defined as a pro-apoptotic molecule that plays an essential role in the mediation of mitochondria-dependent cell apoptosis [13]. To determine the potential involvement of Bid in 5-MCDE-induced cell apoptotic responses, we first evaluated apoptotic induction in cell response to 5-MCDE exposure in wild-type (WT) MEFs. The incubation of MEFs with 5-MCDE for 24 hours resulted in cellular apoptotic responses at doses higher than 0.25 µM in comparison to those of medium control by flow cytometric analysis with PI staining (Fig. 1A). This finding was further confirmed by the induction of cell death by MTT cell viability assay (Fig. 1B) and the induction of cleavages of caspase-3 and PARP, two indicators of apoptosis (Fig. 1C) [14]. This apoptotic induction appeared to be in a dose-dependent manner (Fig. 1). Exposure of MEFs to 5-MCDE at a high dose (0.5µM) caused 44.6% cell death, whereas a low dose (0.25µM) treatment led to marginal cell apoptotic response (Fig. 1A). These results demonstrate that 5-MCDE at the dosages of 0.25–0.5µM enables the apoptotic responses of MEFs.

Fig. (1). 5-MCDE induced cell death in mouse embryonic fibroblasts (MEFs).

A, WT MEFs (3×10⁵) were seeded into each well of 6-well plates and cultured until the cell density reached 80–90% confluence. The cell culture medium was replaced with 0.1% FBS DMEM for 12h. The cells were then exposed to 0–0.5µ M of 5-MCDE for 24 h. The cell death was determined using propidium iodide staining and detected by flow cytometry. AP represents the percentage of apoptotic cells. The histogram was representative of three independent experiments. B, Determination of cell viability using MTT assay. WT MEFs (1 × 10⁴/well) were incubated in different doses of 5-MCDE (0–0.5µ M). After 24 h incubation, cell viability was analyzed by MTT cell viability assay kit. Data are expressed as mean±SEM (n=6). C, WT MEFs were treated with 0–0.5µ M 5-MCDE for 12h, and the inducible cleavages of caspase-3, PARP and Bid were detected by Western blot. GAPDH was used as the protein loading control. Numbers below the blot represent the percentage of cleaved-caspase-3 band intensity relative to the medium control.

It has been previously shown that the BH3-only Bid is important for apoptosis induced by DNA damage treatments [5]. It has also been reported that Bid is predominantly located in the cytoplasm of a cell without treatment. Following TNFα or Fas treatment, Bid is cleaved by caspase-8 to caspase-truncated Bid (tBid), which is required for the release of cytochrome c and subsequent cellular apoptosis. To determine whether Bid is involved in the cell apoptotic response to 5-MCDE exposure, we evaluated Bid cleavage in MEFs treated with 5-MCDE. Consistent with the apoptotic response, the increased tBid was mainly observed in the MEFs exposed to 0.5 µM 5-MCDE (Fig. 1C), suggesting that Bid might be implicated in the cellular apoptotic induction by a relatively high dose of 5-MCDE exposure. To further address this issue, Bid knockout (Bid^−/−) MEFs were employed. The deficiency of Bid protein expression in Bid^−/− cells was identified by Western blot with a specific Bid antibody as compared with parental WT cells (Fig. 2A). The incubation of WT cells with 0.5 µM of 5-MCDE for 24h resulted in significant cell death assayed by MTT and flow cytometric analysis, whereas such cell death was dramatically inhibited in Bid^−/− cells under same experimental conditions (Figs. 2B–2D). These results indicate that genetic knockout of Bid impaired the cellular apoptotic response to high dose of 5-MCDE (0.5 µM) as compared with that in WT MEFs, which is consistent with the Bid biological function demonstrated in the previous studies [15]. However, upon a relatively low dose of 5-MCDE (0.25 µM) exposure, the opposite result was observed in the Bid^−/− MEFs (Figs. 2E–2G). The deficiency of Bid expression led to an increase in cell apoptosis in Bid^−/− MEFs treated with 0.25 µM 5-MCDE as compared with that in WT MEFs (Figs. 2E–2G). Furthermore, the increased 5-MCDE-induced cellular apoptosis in Bid^−/− MEFs could be significantly reduced after reconstituted expression of Bid in the same cells (Figs. 2E–2G), demonstrating that Bid may play a novel anti-apoptotic function in MEFs exposed to low dose (0.25 µM) of 5-MCDE exposure.

Fig. (2). Bid mediated anti-apoptotic response upon low dose of 5-MCDE exposure in MEFs.

A, Identification of Bid expression in the WT and Bid^−/− MEFs. Bid^−/− (stable transfected with the GFP-tagged pcDNA3 vector) and Bid^−/−(GFP-Bid) (stable transfected with the GFP-tagged Bid/pcDNA3 vector) MEFs were analyzed for the GFP-tagged Bid expression by Western blot using antibodies that are specific against GFP and Bid; β-Actin was used as the protein loading control. B and C, Flow cytometric analysis for the apoptosis of WT and Bid^−/− MEFs with or without 5-MCDE (0.5 µM) treatment for 24h. AP represents the percentage of apoptotic cells. D, Determination of cell viability using MTT assay. WT and Bid^−/− MEFs (1 × 10⁴/well) were incubated with or without 5-MCDE (0.5 µM) for 24h. The cell viability was analyzed by an MTT cell viability assay kit. Data are expressed as mean±SEM (n=6). E and F, Flow cytometric analysis for the apoptosis of WT, Bid^−/− (GFP), and Bid^−/−(GFP-Bid) MEFs with or without 5-MCDE (0.25 µM) treatment for 24h. AP represents the percentage of apoptotic cells (E). The histogram is representative of three independent experiments (F). G, The cell viability of WT, Bid^−/−(GFP), and Bid^−/− (GFP-Bid) MEFs with or without 5-MCDE (0.25 µM) treatment for 24h, as measured by MTT assay. Data are expressed as mean±SEM (n=6).

JNKs and p38 Kinase Pathways Were not Involved in Bid-mediated Anti-Apoptotic Response upon 5-MCDE Exposure

JNKs and p38 kinases are two major stress signaling pathways that have been demonstrated to regulate cell apoptotic responses in various experimental systems [16, 17]. To determine whether the Bid-mediated anti-apoptotic effect in MEFs’ response to low doses of 5-MCDE exposure is through the inhibition of either JNKs or p38 kinase pathways, the activation of JNKs and p38 kinase upon 5-MCDE exposure was compared between WT and Bid^−/− MEFs. The results show that the activation of both JNKs and p38 kinase, as determined by Western blot, were increased in similar levels between both types of MEFs, at both time points of 90 mins and 12 hrs after 5-MCDE treatment; and the levels of non-phosphorylated JNKs and p38 kinase were also comparable between both cells (Fig. 3). Consistent with JNKs and p38 kinases, the activation of their downstream transcription factor c-Jun, a major component of AP-1, did not show any difference between WT and Bid^−/− cells (Fig. 3). These results indicate that both kinases and their downstream transcription factor AP-1 are not implicated in Bid-mediated antiapoptotic effect in cells upon 5-MCDE response even though 5-MCDE is able to activate JNKs and p38 kinase.

Fig. (3). The activation of JNKs, p38 and c-jun, were not changed between WT and Bid ^−/− MEFs in cell response to 5-MCDE exposure.

WT and Bid^−/− MEFs were exposed to 0, 0.125 or 0.25 µM of 5-MCDE for 90 min (A) or 12 hrs (B). The activation of JNKs, p38, and c-Jun was detected by Western blot. GAPDH was used as the protein loading control.

Expression of COX-2, but not Bcl-2, was Impaired in Bid^−/− MEFs in Comparison with That in WT MEFs

Since Bcl-2 is able to protect cells from cellular apoptosis [18], we evaluated whether Bcl-2 plays a role in the Bidmediated anti-apoptotic effect in 5-MCDE response. As shown in Fig. (4A), 5-MCDE exposure was able to downregulate Bcl-2 expression in WT MEFs; and the knockout of Bid did not show any observed changes for the downregulation of Bcl-2 expression induced by 5-MCDE in comparison to that in WT MEFs (Fig. 4A), suggesting that Bcl-2 is not involved in the anti-apoptotic effect mediated by Bid upon 5-MCDE exposure.

Fig. (4). Expression of COX-2, but not Bcl-2, was impaired in Bid^−/− MEFs as compared with WT MEFs.

A, WT, Bid^−/− MEFs were exposed to 0, 0.125 or 0.25 µM of 5-MCDE for 24h. The cells were collected and Bcl-2 protein expression was detected by Western blot. GAPDH was used as the protein loading control. B, WT cells were exposed to various doses (0, 0.125, 0.25 or 0.5µM) of 5-MCDE for the indicated time, and the inducible expression of cox-2 mRNA and COX-2 protein were detected by RT-PCR (12h) and Western blot (24h). C, WT and Bid^−/− MEFs were exposed to various doses (0, 0.125, 0.25 or 0.5µM) of 5-MCDE, and the inducible expression of cox-2 mRNA and COX-2 protein were detected by RT-PCR (12 h) and Western blot (12 or 24 h), respectively. GADPH protein loading control in WT cells was the same as in (Fig. (1C)). Numbers below the band represent the percentage of band intensity of cox-2 mRNA relative to the medium control. D, WT, Bid^−/− (GFP) and Bid^−/− (GFP-Bid) MEFs were exposed to 0.25 µM of 5-MCDE, and the inducible expression of cox-2 mRNA (12 h) and COX-2 protein (24 h) were detected, respectively. Numbers below the blot represent the percentage of band intensity of COX-2 relative to the medium control.

Previous studies from various groups have demonstrated that COX-2 could play a role in the protection of stress-exposed cells from undergoing apoptosis in some experiment systems [11, 19]. Therefore, it is of interest to investigate the potential contribution of COX-2 protein expression in cell response to 5-MCDE exposure. Initially we tested whether 5-MCDE is able to increase COX-2 expression in MEFs. The results show that 5-MCDE exposure increased COX-2 expression at both the mRNA and protein levels in a dose-dependent manner (Fig. 4B). Genetic deletion of Bid resulted in dramatically reduction of 5-MCDE-induced cox-2 mRNA levels at all doses tested, and COX-2 protein expression at low doses (≤0.25 µM) in comparison with those in WT MEFs (Fig. 4C), suggesting that Bid is required for 5-MCDE-induced COX-2 expression. It has been noted that the deficiency of Bid expression resulted in the dramatically reduction of cox-2 mRNA level in cell response to 0.5 µM 5-MCDE exposure, whereas the COX-2 protein expression with the same experimental conditions was only partially inhibited (Fig. 4C). The explanation for this may be that 5-MCDE at the dose of 0.5 µM might also cause the COX-2 protein accumulation through modulation of its degradation. To rule out the possibility that the reduction of COX-2 expression might be the cause of other gene mutations which occurred during the establishment of Bid^−/− MEFs, the GFP-tagged Bid/pcDNA3 was used to determine whether reconstituted expression of Bid in Bid^−/− MEF s was able to restore the COX-2 protein expression upon 5-MCDE exposure. After transfection of GFP-Bid/pcDNA3 into Bid^−/− MEFs, Bid-GFP protein expression was detected in Bid^−/− (Bid) as com-pared to that in Bid^−/− (vector) MEF s. As anticipated, the reconstituted expression of GFP-tagged Bid in Bid^−/− MEFs could partially restore the COX-2 induction at both the mRNA and protein levels (Fig. 4D). These data substantially demonstrate that Bid plays an important role in the COX-2 induction in MEFs due to 5-MCDE exposure.

COX-2 Was Required for Bid-Mediated Anti-Apoptotic Effect in Response to Low Dose 5-MCDE Exposure

To identify the contribution of COX-2 protein in the Bid-mediated anti-apoptotic effect in MEFs’ response to low doses of 5-MCDE exposure, COX-2 specific siRNA (si COX-2) was used, which was constructed in our previous study [11]. As shown in Fig. (5A), introduction of siCOX-2 dramatically reduced COX-2 protein expression. The knockdown of COX-2 expression by its siCOX-2 resulted in an increase in cell apoptosis induced by 0.25 µM 5-MCDE indicated in both the PI staining assay and cleavage of caspase-3 assay, as well as MTT cell viability assay (Figs. 5B-5D). These data, together with the findings of the deficiency of COX-2 induction by 5-MCDE in Bid^−/− MEFs, demonstrate that COX-2 is responsible for Bid-mediated anti-apoptotic effect upon low doses of 5-MCDE exposure.

Fig. (5). COX-2 was required for Bid-mediated anti-apoptotic effect in cell response to low dose of 5-MCDE exposure.

A, Identification of COX-2 expression in the WT and its COX-2 siRNA stable transfectants by Western blot. β-Actin was used as the protein loading control. B, Flow cytometric analysis for the apoptosis of WT and its COX-2 siRNA stable transfectants with or without 5-MCDE (0.25 µM) treatment for 24h. AP represents the percentage of apoptotic cells. The histogram was representative of three independent experiments. C, WT and its COX-2 siRNA stable transfectants were treated with 5-MCDE (0.25 µM) for 12h, and the inducible cleavage of caspase-3 was detected by Western blot. β-Actin was used as the protein loading control. D, Determination of cell viability using MTT assay. WT and its COX-2 siRNA stable transfectants cells (1 × 10⁴/well) were incubated with or without 5-MCDE (0.25 µM) for 24h. The cell viability was analyzed by an MTT cell viability assay kit. Data are expressed as mean±SEM (n=6).

Deficiency of Bid Resulted in Abrogation of NFκB Activation in Bid^−/− MEFs

Since the promoter region of cox-2 gene contains two putative binding sites for the transcription factor NFκB [20], and NFκB is an inducible and ubiquitously expressed transcription factor responsible for regulating cell survival [21], we tested whether the IKK/NFκB pathway is a Bid downstream pathway responsible for COX-2 induction by 5-MCDE. The results show that 5-MCDE exposure led to the NFκB transactivation in a dose-dependent manner with the optimal dose of 0.25 µM in NFκB luciferase reporter assay (Fig. 6A), and the significant increases in the NFκB EMSA (Fig. 6B). The differential doses required for the maximal NFκB activation in two assays may be due to the differential apoptosis at different time points after 5-MCDE exposure. The EMSA is to determine the NFκB DNA binding activity that could be detected at 4 hours after 5-MCDE exposure, whereas the NFκB-luciferase reporter transactivation assay is to determine the luciferase activity that could be detected at 12 hours after 5-MCDE exposure. As shown in Fig. (1), 5-MCDE was able to induce cell apoptosis at a dose of 0.5 µM, so there might be some apoptotic induction at 12 hours for the NFκB luciferase assay, whereas this apoptosis does not occur at 4 hours after 5-MCDE exposure. The results from the supershift EMSA indicate that NFκB is activated by 5-MCDE. To determine the potential role of NFκB in Bid-mediated anti-apoptotic effect, the activation of the IKK/NFκB pathway in Bid^−/− MEFs was used. The results show that the knockout of Bid resulted in abrogation of IKKα/β activation (Fig. 6C), NFκB transcriptional activation, as well as DNA binding activity(Fig. 6D), suggesting that Bid is specifically essential for NFκB activation by 5-MCDE. This notion was further supported by the result accomplished by using reconstituted GFP-Bid expression in Bid^−/−. As shown in Fig. (6D), 5-MCDE-induced NFκB binding activity was restored in Bid^−/− (GFP-Bid) as compared with that in Bid^−/− (GFP) MEFs. These data demonstrated that Bid is required for 5-MCDE-induced NFκB activation. Consistent with the important role of Bid in 5-MCDE-induced NFκB activation and COX-2 expression, the genetic knockout of IKKβ (IKKβ^−/−) (Fig. 7A) also impaired COX-2 induction by 5-MCDE (Fig. 7B). Moreover, reconstituted expression of HA-IKKβ was able to restore 5-MCDE-induced COX-2 expression in IKKβ^−/− MEFs (Fig. 7C). These results demonstrate that the IKKβ/NF-κB pathway is a Bid downstream transcription factor responsible for its mediation of COX-2 induction by 5-MCDE.

Fig. (6). Deficiency of Bid resulted in abrogation of NFκβ activation in Bid^−/− MEFs.

A, WT MEFs NFκB-Luc mass1 (8×10³) were seeded into each well of 96-well plates, and the cells were treated with various doses of 5-MCDE in 0.1% FBS DMEM for 12h. The cells were then harvested, extracted with lysis buffer, and the luciferase activity was measured as described in material and methods. The results are expressed as NFκB activity relative to medium control (relative NFκB activity). Each point indicates the mean and standard deviation of triplicate wells. B, WT MEFs were exposed to 0, 0.25 and 0.5µM of 5-MCDE for 4h. The cells were collected, and the extracted nuclear protein for NF κB DNA binding activity was measured individually by Electromobility shift assay (EMSA) as described in material and methods. C, WT and Bid^−/− MEFs (3×10⁵) were exposed to various concentrations of 5-MCDE for 180 minutes. The cell extracts were analyzed by Western blot with specific antibodies against P-IKKα/β, IKKβ, and IKKα β-Actin was used as the protein loading control. Numbers below the blot represent the percentage of P-IKKα/β band intensity relative to the medium control. D, WT, Bid^−/− and Bid^−/− (Bid) MEFs were treated with 0.25 µM of 5-MCDE for 4h. The cells were collected, and the extracted nuclear protein for NFκB DNA binding activity was measured individually by EMSA as described in material and methods.

Fig. (7). The IKKβ/NF-κB pathway is responsible for Bid mediation of COX-2 induction by 5-MCDE.

A, WT/vector control transfectant, IKKβ^−/−, and IKKβ^−/− (IKKβ) transfectants were harvested and extracted. The cell extracts were analyzed by Western blot with specific antibodies as described above. β-Actin was used as a protein loading control. B, WT and IKKβ^−/− MEFs were treated with various doses of 5-MCDE for 24 h, and the inducible expression of COX-2 protein was detected by Western blot. β-Actin was used as a protein loading control. C, WT, IKKβ^−/− and IKKβ^−/− (IKKβ) MEFs were treated with different doses of 5-MCDE for 24 h, and the inducible expression of COX-2 protein was detected by Western blot. β-Actin was used as a protein loading control.

DISCUSSION

Bid has been well characterized as a mediator for mitochondrial-dependent cell apoptosis [13]. Our current studies provide direct evidence demonstrating its novel dual function of the regulation of cell apoptosis in MEFs response to 5-MCDE exposure. We found that genetic knockout of Bid in MEFs led to the dramatic inhibition of cell apoptosis in MEFs’ response to a relatively high-dose (>0.5 µM) of 5-MCDE, whereas it increased MEFs apoptotic response to a relatively low-dose (0.25 µM) of 5-MCDE.

In multi-cellular organisms, homeostasis is maintained through a balance between cell proliferation and cell death [22]. Apoptosis is a tightly regulated mechanism in living organisms to eliminate redundant, damaged, or infected cells. The inhibition of normal apoptotic responses may cause an imbalance of normal tissue homeostasis, which may promote cell growth and allow the survival of genetically damaged cells, which subsequently contributes to carcino-genesis [23]. Apoptosis is orchestrated by pro- and antiapoptotic factors, whose balance determines the fate of the distressed cells. The execution of apoptosis can occur via extrinsic and intrinsic distinct signaling pathways. The extrinsic pathway is initiated by the binding of apoptosis-inducing ligands to cell surface death receptors associated with Fas-associated death domain (FADD) [24]. The intrinsic pathway is initiated by intercellular stress, lack of growth factors, or overwhelming DNA injury which subsequently targets the mitochondrial membrane [25]. Loss of mitochondrial membrane potential and increased membrane permeability lead to the release of cytochrome c [26]. The release of this protein into the cytosol results in the activation of apoptotic protease activating factor-1 (APAF-1) and caspase-9 recruitment [25]. These proteins form a functional apoptosome that activates the effector caspase cascade, again resulting in apoptosis [24]. While these two pathways are often described as separate pathways, significant cross-talk is thought to exist. Our current studies show that Bid mediates apoptotic response in MEFs treated with relatively high doses of 5-MCDE exposure, whereas it may also be implicated in the protection of MEFs from the apoptosis in cell response to relative low dose of 5-MCDE exposure. This dual function of Bid in cell response to 5-MCDE exposure provides a novel scenario for the understanding the crosstalk among various cell survival and apoptotic pathways via different upstream signaling pathways even thought this occurs through a single same protein. Although this finding causes us to be cautious with regard to the rational that the proposed use of Bid activator in combination with other anticancer drugs in clinical applications, it also provides an alternative for targeting Bid for cancer therapy and chemoprevention. The dual functions of Bid have also been demonstrated in vivo studies. It has been reported that deletion of Bid inhibits carcinogenesis following the administration of a chemical carcinogen, diethylnitrosamine, in the liver, although this genetic alteration promotes tumorigenesis in the myeloid cells [4], suggesting that the role of Bid in carcinogenesis in vivo is organ- and/or etiology-specific [4]. There- fore, further evaluation of anti-apoptotic effect of Bid in vivo carcinogenesis model will be a very interesting goal for our future studies. Although we are far from understanding the mechanisms of the involvement of Bid in the balance of cell death and survival, we anticipate that Bid may be a unique and fascinating molecule switch in the determination of cell fate. Further comparison of mitochondria functions and other detailed molecular mechanisms involved in its function in apoptosis and anti-apoptosis in the cell response to various doses of 5-MCDE might provide a clearer understanding of the regulation of cell death and survival pathways. Fully understanding the nature of Bid in the regulation of cell death and survival both in vitro and in vivo will provide crucial information for our potential utilization of Bid as a target for cancer prevention and therapy.

The role of Bid in the mediation of cell apoptosis could be generally considered as the bridge connecting various peripheral death pathways to the mitochondria, which then initiate or amplify the apoptosis biochemical machinery [4]. Thus, Bid is initially defined as a pro-death molecule and a sentinel of intracellular damage. NFκB plays a critical role in the regulation of cell apoptosis by modulation of its downstream cell survival gene expression [27]. Our results indicate that NFκB activation and DNA binding activity are increased in the WT cells exposed to 5-MCDE in a dose-dependent manner. 5-MCDE-induced IKKβ phosphorylation and NFκB-dependent transcriptional activity as well as DNA binding activity are substantially reduced in Bid^−/− MEFs, which could be restored through the reconstituted introduction of GFP-Bid. NF-κB is known to regulate COX-2 expression [27], which has been demonstrated to play a critical role in the protection of cells from apoptosis in our previous studies [12]. Consistent with NFκB activation, COX-2 expression in Bid^−/− MEF s is abolished in comparison with that in WT MEFs. Our studies also indicate that the IKKβ/NFκB pathway is responsible for 5-MCDE-induced COX-2 expression and this COX-2 induction by 5-MCDE mediates the anti-apoptotic effects of Bid. Therefore, the current studies demonstrate a novel function of Bid in the regulation of cell apoptosis via mediation of NFκB activation. The molecular mechanism linking Bid to NFκB activation is a very interesting question for fully understanding of function of Bid in cellular apoptotic responses. Previous studies have demonstrated that activated Bid mainly translocates into mitochondria and activated NF-κB translocates into nuclear. We therefore anticipate that Bid-mediated IKKβ/NF-κB activation might not be through direct protein-protein interaction. Considering that reactive oxygen species (ROS) is mostly generated in mitochondria and is a major mediator for NFκB activation [28], we further anticipate that 5-MCDE-induced Bid activation may be involved in the regulation of ROS generation, and subsequently leading to the activation of IKKβ/NF-κB pathway. This hypothesis is under investigation in our laboratory.

Previous studies demonstrate that caspase-8 is activated from the cleavage of procaspase-8, which eventually cleaves Bid into a p15 form tBid. tBid then translocates into mitochondria, where it activates oligomerization of Bak or Bax and activates the apoptotic cascades of caspase-9 and caspase-3, which leads to cell apoptosis [4]. Our current studies only show that tBid generation are majorly observed in the MEFs exposed to relative high dose 5-MCDE (0.5 µM), while low doses of 5-MCDE (≤0.5 µM) could not induce observable tBid generation (Fig. 1C), suggesting that tBid might not be implicated in anti-apoptotic function of Bid due to low doses of 5-MCDE exposure.

It is accepted that under environmental stress, the cell fate is dependent on the balance of both apoptotic pathways and cellular survival pathways. The IKKβ/NF-κB signaling pathway transmits signals that are essential for cell survival in a variety of physiologic and pathological processes [12, 29], while JNK and p38 kinase, as well as Bid are well-known cell apoptotic pathways of cellular response to high doses of environmental stress [10, 30]. Also, the roles of the IKKβVNF-κB pathway and the JNK pathway in cellular death decision are dependent on cell-types and stress conditions [10]. Therefore, this may be a reasonable explanation for our observation of current studies where exposure of MEFs to a relatively high dose of 5-MCDE (0.5 µM) increased both cell apoptosis and NF-κB activation (Figs. 1 and 6). We anticipate that the activation of cell apoptotic pathway due to 0.5 µM 5-MCDE exposure might be dominant, although the anti-apoptotic IKKβ/NF-κB pathway is also activated under same 5-MCDE exposure.

In summary, the current study demonstrates a novel function of Bid in the activation of the IKKβ/NFκB signaling pathway, by which Bid induces COX-2 expression and in turn protects the MEFs from apoptosis in cell response to relatively low doses of 5-MCDE exposure. Although the molecular mechanisms of Bid-activated NFκB are not presently clear, Bid-mediated generation of reactive oxygen species [31] is anticipated to be involved. Further studies of the mechanisms involved in the Bid-mediated NFκB activation will help us to understand the nature of Bid biological function in the regulation of cell apoptosis.

ABBREVIATIONS

AP-1

activator protein-1

COX-2

cyclooxygenase-2

DMEM

Dulbecco's modified Eagle's medium

FBS

fetal bovine serum

IKK

IκB kinase

JNKs

c-Jun N-terminal kinases

MAPKs

mitogen activating protein kinases

5-MCDE

(±)-anti-5-methylchrysene-1,2-diol-3,4-epoxide

MEFs

mouse embryonic fibroblasts

NF-κB

nuclear factor-kappaB

PARP

poly (ADP-ribose) polymerase

WT

wild-type