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4  Results

4.1 Transcriptional Regulation of 15-LOX-1 expression

4.1.1 Induction of 15-LOX-1 expression in A549 human lung epithelial cells

A549 cells were cultured for various time periods in the presence of 670 pM IL-4. The cells were harvested and the lysates were analysed for the expression of 15-LOX-1 mRNA by RT-PCR using 15-LOX-1 specific primers. 15-LOX-1 mRNA was first observed after minimum 12 h of IL-4 stimulation. The highest mRNA concentration was detected after a 24 h incubation period (Fig. 7A). After longer incubation periods the mRNA levels dropped perceptibly. To find out whether IL-4 has to be present during the entire incubation period or whether a single cytokine stimulus may be sufficient to induce 15-LOX-1 expression the following experiment was carried out. A549 cells were exposed to IL-4 for various time periods, the cytokine was washed away and incubation was resumed for a total of 24 h. Finally, the expression of 15-LOX-1 mRNA was analysed by RT-PCR.

Figure 7. Induction of 15-LOX-1 mRNA was delayed and requires continuous exposure to IL-4.

Panel A: A549 cells were incubated with 670 pM IL-4 in serum-free medium for different periods (0-72 h), the cells were harvested, and total RNA was extracted. Semiquantitative RT-PCR was performed to detect 15-LOX-1. β-Actin RT-PCR was used for the normalization of 15-LOX-1 expression. Panel B: A549 cells were exposed to IL-4 (670 pM) for varying periods (0-11 h). IL-4 was removed by washing thrice with PBS, and incubation was resumed for a total of 24 h; then the cells were lysed for RNA extraction. 15-LOX-1 expression was assayed by RT-PCR.

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As shown in Fig. 7B, 15-LOX-1 expression in A549 cells required a minimum of 11 hours continuous exposure to IL-4. These data indicate that a single IL-4 stimulus was not sufficient to up-regulate expression of the 15-LOX-1 mRNA.

4.1.2 Activation of 15-LOX-1 mRNA expression does not require de novo protein synthesis

As a time lag of 12 h was observed in the start of transcription, it was probable that de novo synthesis of additional regulatory factors was required. To test this hypothesis stimulation with IL-4 was performed in the presence of cycloheximide (10 µg/ml), a protein synthesis inhibitor and the presence of 15-LOX-1 mRNA detected by PCR. As shown in Fig. 8. cycloheximide did not affect the activation of 15-LOX-1

Figure 8 De novo protein synthesis was not required for induction of 15-LOX-1 mRNA

Cells were exposed to cycloheximide (10 µg/ml) along with IL-4 (670 pM) for 12 h. 15-LOX-1 expression was assayed by RT-PCR.

4.1.3 IL-4 upregulates histone acetyltransferases in A549 cells

Activation of cellular acetyltransferases may constitute an additional regulatory element in the intracellular signal transduction cascade (Kouzarides, 2000). Acetylation of histones causes conformational changes of nuclear proteins leading to demasking of potential transcription factor binding sites, so that the transcription factor may bind to the promoter of target genes. Since histone acetylation has recently been implicated in the induction of 15-LOX-1 expression in CaCo-2 cells (Kamitani et al., 2001),the effect of IL-4 on HAT activity was tested in A549 cells. For this purpose, cells were exposed to IL-4 (670 pM) for 3 h and the cell lysates were assayed for HAT activity. IL-4 significantly upregulated HAT activity even after a relativly short incubation periods (Fig. 9). The HAT activity is the sum of several catalytic processes and involves the activity of various proteins. One of these enzymes is the transactivating protein CBP/p300, which exhibits a strong HAT activity. To find out whether or not CBP/p300 was involved in IL-4 induced upregulation of acetyltransferase activity in [page 42↓]A549 cells, the cells were transfected with the viral oncoprotein wtE1A, which has been identified as an endogenous inhibitor of CBP/p300.

After IL-4 treatment the transfected cells exhibited a significantly reduced HAT activity (Fig. 9). However, in cells transfected with E1AmCBP, a mutant of E1A protein incapable of binding to CBP/p300, no increase in IL-4 induced HAT activity was observed. These data indicate that the augmented acetyltransferase activity was mainly due to activation of CBP/p300.

Figure 9 IL-4 uprgulates histone acetyl transferase activity via CBP/p300:

A549 cells were exposed to IL-4 for 3 h, and cell lysates were used to measure the histone acetyltransferase activity. Cells were either untransfected or transfected with the wtE1A or with its antagonizing mutant E1AmCBP. The experiments were repeated at least thrice (n = 4).

4.1.4 HDACs stimulate IL-4 induced 15-LOX-1 expression

The degree of acetylation of nuclear histones depends on cellular HAT activity but also on the activity state of histone deacetylases (HDACs). In resting cells there appears to be a steady state of acetylating and deacetylating events and inhibitors or activators of either process may shift the equilibrium in either direction. Recently, it has been reported that activation of HDAC may cause alterations in the chromatin state and may inhibit gene transcription (Kao et al., 2000). If the hypothesis that CBP/p300 catalyzed histone acetylation is important for IL-4 induced 15-LOX-1 expression was true, then inhibitors of cellular HDAC are likely to act synergistically to IL-4 or may even be capable of inducing 15-LOX-1 expression in the [page 43↓]absence of IL-4. To test this conclusion A549 cells were incubated with suboptimal doses (335 pM) of IL-4 in the presence of sodium butyrate (NaBT) and the expression of 15-LOX was assayed by Western blotting.

Figure 10 IL-4 synergestically upregulates the expression of 15-LOX-1expression by sodium butyrate.

Cells were cultured for 24 h in the presence of a suboptimal dose of IL-4 (330 pM) alone, 2 mM sodium butyrate (NaBT) alone, or of both together. The cells lysates were then analyzed for 15-LOX-1 expression using a specific antibody. The amount of protein was normalized by Western blotting with β-actin antibody.

From Fig. 10. it can be seen that IL-4 at suboptimal concentrations did not induce 15-LOX-1 expression. However, in the presence of NaBT a strong LOX signal was observed. Interestingly, NaBT did also induce 15-LOX-1 expression in the absence of IL-4 albeit a weaker signal was detected. Similar results have recently been reported for other cellular systems (Kamitani et al., 2001).

4.1.5 In vitro binding of transcription factors and the role of tyrosine phosphorylation

Since histone acetylation may be important for binding of transcription factors to the promoter of the 15-LOX-1 gene, in vitro transcription factor binding assays were carried out. A549 cells were incubated with IL-4 for different time periods. Nuclear extracts were prepared and binding studies of nuclear proteins to the 15-LOX-1 promoter were performed. In cells, which were cultured in the absence of IL-4, no promoter binding proteins were detected (Fig. 12, lane 1). In contrast, a variety of 15-LOX-1 promoter-binding proteins were present in the nucleus of IL-4-treated cells (Fig. 12, lane 2 and 3).

Figure 11 Tyrosine phosphorylation was essential for STAT6 activation.

A549 cells were treated with IL-4 for 0, 1and 6 h or pretreated with genistein. Invitro binding assays were performed with the nuclear extracts and 15-LOX-1 promoter. The proteins bound to the promoter were assayed by Western blotting for the presence of STAT6.

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Figure 12 In vitro binding of transcription factors was dependent on tyrosine phosphorylation.

A549 cells were cultured for 1 and 12 h in the absence or presence of IL-4 (670 pM). Before starting the incubation, one batch of cells was pretreated with genistein (25 µg/ml) for 15 min. After the incubation period, cells were harvested, nuclear extracts were prepared, and DNA binding assays were performed. First lane, cells incubated for 12 h in the absence of IL-4; second lane, cells incubated for 1 h in the presence of IL-4; third lane, cells incubated for 12 h in the presence of IL-4; fourth lane, genistein-pretreated cells incubated for 12 h in the presence of IL-4.

As expected STAT6 was one of the major components and its identity was confirmed by Western blots using commercially available anti-STAT6 antibodies (fig. 11). Interestingly, the pattern of the binding proteins was very similar when cells were treated with IL-4 for 1 or 12 hours (Fig. 11, lane 2 and 3). These data indicate that under in vitro conditions the transcription factors including STAT6 are capable of binding to the immobilized 15-LOX-1 promoter and that 1 h incubation was sufficient for maximal in vitro binding. Combining these data (rapid in vitro binding) with the results shown in Fig. 7 (delayed 15-LOX-1 expression) one may conclude that in vivo the binding of phosphorylated STAT6 to the promoter was hindered. Alternatively, co-activators exhibiting a prolonged time-dependence may be required for transcriptional regulation of the 15-LOX-1 gene.

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It has been reported for other cell types that tyrosine phosphorylation was involved in IL-4 and IL-13 induced 15-LOX-1 expression (Conrad and Lu, 2000). Thus, the effect of genistein, a potent tyrosine kinase inhibitor, on protein phosphorylation and on the binding activity of nuclear proteins to the 15-LOX-1 promoter was examined. A549 cells were treated with genistein (25 µg/ml) for 30 min. After washing away the inhibitor 670 pM IL-4 was added and the cells were cultured for additional 12 hours. Subsequently, the nuclear extracts were analyzed for the presence of 15-LOX-1 promoter binding proteins. From Fig. 12 (lane 4) it can be seen that genistein completely blocked the binding of proteins to the promoter and abrogated 15-LOX-1 expression (Fig. 13). Similarly treated cells were also analysed for the presence of acetylated STAT6. Genistein was observed to inhibit IL-4 induced acetylation of STAT6 (Fig. 13).

Figure 13 Tyrosine phosphorylation of STAT6 was important for 15-LOX-1 expression.

Cells were treated with IL-4 for 12 h with or without preincubation with genistein. The proteins were assayed for acetyl STAT6 and 15-LOX-1 by Western blotting.

These data suggests that tyrosine phosphorylation in A549 cells was a prerequisite for STAT6 acetylation and binding to the 15-LOX-1 promoter.

4.1.6 In vivo binding of STAT6 and histones to the 15-LOX-1 promoter

From Fig. 12, it was concluded that under in vitro conditions phosphorylated STAT6 was capable of binding to the 15-LOX-1 promoter. The next series of experiments were aimed at addressing the question of whether or not such a binding may actually occur in vivo. For this purpose A549 cells were cultured in the presence of IL-4 for various time periods and DNA binding proteins were cross-linked to the nucleic acid by formaldehyde treatment. Then STAT6 was immunoprecipitated with a specific antibody and the cross-linked DNA was [page 46↓]analysed by PCR using 15-LOX-1 promoter specific primers. We found that the earliest binding of STAT6 was detected after 11 hours of IL-4 exposure (Fig. 14). These data were somewhat surprising since both STAT6 phosphorylation and its in vitro binding were rapid processes. Thus, it was concluded that the binding of STAT6 to the 15-LOX-1 promoter in vivo was hindered during early phases of the incubation period.

Figure 14 Differential kinetics of in vivo binding of STAT6, histone, and acetylated histone to the 15-LOX-1 promoter.

A549 cells were exposed to IL-4 (670 pM) for the periods indicated and then treated with formaldehyde to cross-link DNA binding proteins to the DNA. The protein-nucleic acid complexes were immunoprecipitated with anti-STAT6, anti-histone H3, and anti-acetylhistone H3 antibodies. The cross-linked DNA was purified and analyzed by PCR for the presence of 15-LOX-1 promoter DNA. An aliquot of the complexes was removed before the immunoprecipitations and was similarly processed and used as a control for the PCR reaction. This DNA was referred to as input chromatin.

Similar in vivo binding studies were performed using anti-histone H3 (middle lane) and anti-acetylhistone H3 antibodies (lower lane). It was observed that non-acetylated histones were bound at the early phase of the induction process. In contrast, STAT6 and acetylated histones were bound exclusively at later stages. These data indicate an inverse correlation between the binding of non-acetylated histone and the activation of 15-LOX-1 gene.

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4.1.7  STAT6 and histones physically interact in the acetylated form

Immunoprecipitations were performed to obtain experimental evidence for a physical interaction between STAT6 and histones upon IL4 stimulation. To check for in vivo interaction, the cells were treated with 1% formaldehyde to crosslink the proteins and then immunoprecipitation was performed on the protein extract and then a dual immunoprecipitation strategy was carried out. After the first immunoprecipitation with the anti-acetylhistone H3 antibody the protein was divided into 2 lots. The majority (75%) was used for the second round of immunoprecipitation with STAT6 antibody and western blotting using an anti-acetyl antibody as probe. This laborious method had to be applied since no antibody against acetyl STAT6 was currently available. For immunostaining of the acetylhistone H3 only 25% of the initial immunoprecipitate was used because of its abundance in the cell. This strategy as well as the different cross-reactivity of the anti-acetyl antibody with different acetylated proteins does not allow a direct comparison of the relative amounts of STAT6 and histones H3. At 11 hours after IL4 stimulation, acetyl STAT6 was bound to the acetyl histones while at 4 hours very little of this interaction was observed (Fig. 15).

Figure 15 Interaction between acetyl STAT6 and acetyl histones.

Cells were exposed to IL-4 for 4 and 11 h, and DNA-binding proteins were cross-linked to the DNA by formaldehyde treatment. Cross-linking was reversed and the proteins were analysed by Western blotting for acetylhistone H3 and acetyl STAT6.

This observation was in accordance with the data obtained from the chromatin immunoprecipitaion experiments (Fig. 14). Further, after the reversal of crosslinking, the same immunoprecipitaion was performed with STAT6 antibody and similar results were obtained. Here again, an increased binding of acetyl STAT6 to acetylhistones H3 during the time course of IL-4 treatment was observed (Fig. 16, left panel).

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Figure 16 Kinetics of interaction between STAT6 and histone.

Cells were incubated with IL-4 for 4 and 11 h, and proteins were cross-linked with formaldehyde. The proteins were then immunoprecipitated with either Acetyl Histone H3 or STAT6 antibodies. After the reversal of crosslinking, the proteins were analysed by Westrern blotting for STAT6, acetyl STAT6 and acetyl histone H3.

When immunoprecipitaion was carried out with a STAT6 antibody, no co-precipitation of acetylhistones H3 at 4 h of IL-4 exposure but a strong signal after 11 h was observed (Fig. 16, right panel). The chromatin immunoprecipitation experiments (Fig. 14) showed that after 11 hours of IL-4 exposure only acetylated histones H3 and acetylSTAT6 are bound at the 15 LOX-1 promoter and the increased binding of acetyl histone to STAT6 during the time course of IL-4 exposure (Fig. 15) was in line with these data.

4.1.8 CBP/p300 plays an important role in the acetylation of STAT6

If acetylation of STAT6 and of nuclear histones was crucial for IL-4 induced transcription of the 15-LOX-1 gene, inhibition of acetylation was expected to block this process. It is known from the literature that CBP/p300, a transactivating protein with HAT activity, is capable of acetylating STAT6 (McDonald and Reich, 1999). To study the role of CBP/p300, A549 cells were transfected with wtE1A and one E1A mutant, which differed from the wild-type with respect to its functional properties. The mutant E1AmCBP, which lacks amino acids 64-68 - the CBP binding region - acts as E1A antagonist and does not inhibit the acetyltransferase activity of CBP/p300 (Hecht et al., 2000). After transient transfection of A549 cells with the appropriate cDNA constructs the cells were treated with IL-4 for 12 h. Cells were harvested, lysed and acetylation of STAT6 was measured by Western-blotting using an acetyl-specific antibody (Fig. 17).

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Figure 17 CBP/p300 acetylates STAT6.

Cells were transfected with either wtE1A, E1AmRb or E1AmCBP vector. Cells were then treated with IL-4 for 12 h and protein was analysed by Western blot for acetyl STAT6 protein.

In cells transfected with wtE1A there was no enhancement in STAT6 acetylation. In contrast, cells transfected with the E1AmCBP mutant (E1A antagonist) showed a strong STAT6 acetylation signal.

4.1.9 Acetylation of STAT6 is essential for its promoter binding

The data shown in the previous figures indicate that STAT6 acetylation was upregulated when the cells are stimulated with IL-4 and that the acetyltransferase activity of CBP/p300 was involved. To find out whether STAT6 acetylation was required for its binding to the 15-LOX-1 promoter EMSA were carried out. Cells transfected with wtE1A and with the non-inhibitory mutant E1AmCBP were cultured in the presence of IL-4 for 12 h. The cells were then lysed and EMSA was carried out with STAT6 binding consensus sequences. In Fig. 18 it can be seen that STAT6 binding occurred when cells were treated with IL-4 and similar results were obtained when cells were transfected with the E1A antagonist E1AmCBP, which cannot bind CBP/p300 and therefore was unable to inhibit the CBP/p300 acetylase activity. On the other hand, no STAT6 binding was observed when acetylation was prevented by transfecting the cells with wtE1A.

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Figure 18 Acetylation of STAT6 was a prerequisite for its promoter binding.

EMSA was performed with the protein extracts from the wtE1A and E1AmCBP transfected cells and STAT6 binding element obtained from the 15-LOX-1 promoter.

4.1.10 CBP/p300 is required for 15-LOX-1 expression

The experiments so far have pointed to the importance of CBP/p300 in IL-4 stimulated 15-LOX-1 expression. To confirm this effect, different amounts of wt E1A cDNA were transfected in A549 cells and the cells were stimulated with IL-4. The suppression of 15-LOX-1 expression was found to be dose-dependent by both immunoblotting (Fig. 19a) and activity assays (Fig. 19b).

Figure 19a CBP/p300 was essential for 15-LOX-1 expression.

Dose dependent inhibition of 15-LOX-1 by wtE1A was performed by transfecting cells with varying amounts of wtE1A (0-1.5 µg) and probing the cell lysates with anti 15-LOX-1 antibody.

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Figure 19b CBP/p300 was essential for 15-LOX-1 expression.

15 lipoxygenase activity was determined in the cells by HPLC after treatment with IL-4 for 24h. The cell lysates were incubated with AA and lipids were extracted using acidified ethyl acetate. 13-HODE was used as an internal standard.

For the latter experiment, cells were transfected with wtE1A and E1AmCBP vectors and induced with IL-4 for 24 hours. The cell lysate was then incubated with AA, the products were extracted and HPLC was performed. Amount of 15-HETE formed was quantitated with 13-HODE as internal standard. Analysing the 15-LOX activity as a measure for expression of the functional protein, similar amounts of hydroxy fatty acids were observed in control cells and in cells transfected with E1AmCBP (Fig. 19b). In contrast, a significantly reduced 15-LOX-1 activity was observed when the cells were transfected with wtE1A.

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4.2  Induction of apoptosis by 15-LOX-1

4.2.1 IL-4 causes apoptosis in A549 cells

15-LOX-1 is a lipid peroxidising enzyme and its expression may cause an increase in the cellular concentration of reactive oxygen species and may lead to cell death. To see if IL-4 stimulation had deleterious effects, A549 cells were examined for apoptosis. TUNEL assay showed that IL-4 (670 pM) induces apoptosis in A549 cells after 72 h of stimulation (Fig. 20a).

Figure 20a IL-4 causes apoptosis in A549 cells.

TUNEL assay was performed after incubating A549 cells with IL-4 for 72 h. The right panel shows apoptotic cells stained blue. The left panel is the untreated cells.

Figure 20b IL-4 causes apoptosis inA549 cells.

A549 Cells were treated with IL-4 for 72 h and stained with annexin V-fluos. Only the apoptotic cells exhibited fluorescence. The control cells (not treated with IL-4) did not show any fluorescent staining.

Annexin V staining showed similar results (fig. 20b). During the early stages of apoptosis, cells lose their phospholipid membrane asymmetry and expose phosphatidylserine at the cell surface while the overall structure of the plasma membrane remains intact. Annexin V binds specifically to phosphatidylserine but cannot penetrate the cell membrane. The exposure of phosphatidylserine on the outside of the cell can be monitored using fluorochrome labelled Annexin V. The green fluorescent staining of the cell indicates apoptosis.

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4.2.2  IL-4 induces caspase-3 activation via 15-LOX-1

Next was investigated whether or not caspase-3, a downstream caspase involved in the effector phase of apoptosis, is activated when A549 cells were stimulated by IL-4. The activity of caspase-3 using DEVD-pNA as substrate showed an increase upon IL-4 stimuation and thus confirmed the above findings (Fig. 21). NDGA, a general inhibitor of lipoxygenases was used at concentration of 10 µM to verify the effect of 15-LOX-1. A drastic reduction in the caspase-3 activity was measured in NDGA treated cells. Furthermore, the A549 cells were treated with 15-HETE (product of 15-LOX-1). 15-HETE (30 µM) produced a similar increase in caspase-3 activity as IL-4. Treatment of cells with NDGA before 15-HETE incubation did not show any effect on the caspase-3 activity confirming that the product of 15-LOX-1 was required for caspase-3 activation.

Figure 21 IL-4 induces Caspase-3 activity via 15-LOX-1.

Caspase-3 activity was measured in A549 cells after incubation with IL-4 and 15-HETE. NDGA was used as a lipoxygenase inhibitor to assess the effect of 15-LOX-1 in this process. (n = 3).

4.2.3 IL-4 induced apoptosis was mediated by 15-LOX-1

Apoptosis in A549 cells was measured using Cell Death ELISA. This method assays the presence of DNA-histone nucleosomal complexes in the cytoplasm, which is an indicator of [page 54↓]apoptosis. A549 cells were treated with IL-4 for 72 h, cells were treated with 15-HETE or a similar incubation was performed after pre-treatment with NDGA.

Figure 22 IL-4 induced apoptosis was mediated by 15-HETE and caspase-3.

A549 cells were treated with IL-4, 15-HETE or 15-PGJ2 and amount of apoptosis was measured by Cell Death ELISA. NDGA was used to inhibit the 15-LOX-1 enzyme. (n = 3).

Figure 22 shows the relative increase in the amount of nucleosomal complexes present in the cytoplasm. 15-HETE and IL-4 induced a drastic increase in the amount of apotosis, which was inhibited by NDGA pre-treatment. The cells pre-incubated with a peptide inhibitor of caspase-3, Z-VAD-FMK (100 µM) also showed complete inhibition of apoptosis confirming the central role of caspase-3 in this apoptotic process.

Similar experiments were performed with Beas-2b cells (fig.23), which represent normal tracheobronchial cells. Identical effects were observed in these cells suggesting the process of apoptosis by IL-4 was common in both carcinoma and normal cells.

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Figure 23 IL-4 causes apoptosis in normal tracheobronchial cells.

Normal bronchial cells were treated with IL-4 and the apoptosis was assayed using Cell Death ELISA. (n = 3).

These experiments show the pivotal role of 15-LOX-1 and its product 15-HETE in IL-4 induced apoptosis in lung cells.

4.2.4 15-HETE is a ligand for PPAR γin IL-4-stimulated A549 cells

IL-4 treatment of A549 cells results in the upregulation of 15-lipoxygenase, which in turn augmented the production of 15-HETE. 12/15 lipoxygenase products have earlier been shown to act as ligands for PPARγ at relatively high concentrations. To test the hypothesis that 15-HETE could serve as a ligand for PPARγ in this cell system, A549 cells were labelled with [14C]-arachidonic acid and treated with 670 pM of IL-4 for 72 hours. Total protein extracts prepared from the cells were subjected to immunoprecipitation with PPARγ -antibody and protein-A agarose. The lipids were extracted from the immune complex and analysed by [page 56↓]TLC. In IL-4 treated cells alone a radioactive lipid was observed. It was identified as 15-HETE by radio-TLC by co-chromatography of standard 15-HETE (Fig. 24).

Figure 24 15-HETE acts as a ligand for PPARγ during IL-4 treatment of A549 cells.

A549 cells radioactively labelled with 14C-AA and were treated with IL-4 and the cell lysates were subjected to immunoprecipitation with PPARγ antibody. The lipids were extracted from the immunoprecipitates and subjected to TLC to identify the ligands for PPARγ.

Untreated cells or cells treated with NDGA (10µM) prior to IL-4 challenge failed to show any radioactive ligand for PPARγ. Thus, 15-HETE produced in A549 cells upon IL-4 stimulation acts as a ligand for PPARγ.

4.2.5 IL-4 activates PPARγ via 15-HETE

PPARγ is a nuclear receptor, which upon binding to the ligand gets activated and along with another protein RxR binds to a specified sequence element, PPRE, on the promoter of downstream genes. Binding of the ligand to the receptor is not exclusive to the activation of [page 57↓]the downstream targets of the receptor. Thus, ability of 15-HETE to activate PPARγ downstream genes was tested utilising a PPARγ driven promoter and luciferase reporter.

Figure 25 15-HETE produced in A549 cells upon IL-4 treatment causes an increase in PPARγ dependent promoter activity.

PPARγ dependent promoter activation was in A549 cells after transient transfection with a luciferase reporter vector driven by a PPARγ binding element. The transfection efficiency was normalised by co-transfection with a CMV driven β-galactosidase control vector. (n = 3).

IL-4 increased the PPARγ dependent promoter activity (Fig. 25), which was inhibited by 10 µM NDGA indicating that the enzymatic product of 15-LOX-1 was essential for the promoter activation. 15-HETE (30 µM) was also observed to increase PPARγ dependent promoter activity, confirming the functional interaction between 15-HETE and PPARγ receptors. A similar increase in the luciferase activity was observed with 5 µM 15-deoxy-Δ12,14 PGJ2 (15-PGJ2), a naturally occurring ligand of PPARγ.

4.2.6 IL-4-induced apoptosis was mediated by PPARγ

Cell Death Detection ELISA detects cytoplasmic DNA-histone complexes indicating apoptosis. IL-4 treated cells showed a significant degree of apoptosis as compared to the untreated controls. 15-HETE (30µM) and 5 µM 15-PGJ2, a PPARγ ligand. also caused [page 58↓]equivalent percentage of apoptosis when treated for 72 hours. Cells were transiently transfected with a dominant negative vector for PPARγ and treated with IL-4. In the PPARγ dominant negative vector, PPARγ was leu468 was mutated to Ala and Glu471 was mutated to Ala (Adams et al., 1997), which prevented the ability to activate transcription. PPARγ dominant negative vector abolished the induction of apoptosis by IL-4 (Fig. 26).

Figure 26 IL-4 induced apoptosis via PPARγ.

A549 cells were transiently transfected with PPARγ dominant negative vector (PPARγDN) and treated with IL-4. Apoptosis was measured by Cell Death ELISA and caspase-3 activity assays. (n = 4).

Upon treatment of dominant negative cells with 15-PGJ2 no signs of apoptosis were observed. This indicated that IL-4 induced apoptosis occurs via PPARγ. Similar results were observed with caspase-3 activation too (fig. 26).

4.2.7 PPARγupregulates cleavage of caspase-8

Caspase-8 is one of the important upstream factors involved in the upregulation of caspase-3 activity. Caspase-8 exists as an inactive 54 kDa molecule which is autocleaved into active p41/42 and p18 molecules. A549 cells treated with IL-4 showed significantly higher levels of the cleaved products as compared to the untreated cells and cells treated with 10 µM NDGA [page 59↓]prior to IL-4 induction (Fig. 27). Similar upregulation of cleavage of caspase-8 was observed with 5 µM of 15-PGJ2 and 30 µM 15-HETE.

Figure 27 IL-4 causes cleavage of caspase-8 in A549 cells.

A549 cells were treated with either IL-4, 15-PGJ2 or 15-HETE. The cell lysates were analysed by Western blotting for the cleavage of caspase-8. The major cleavage product is 41 kDa while the minor product is 20 Kda, which was too faint to be detected with this antibody.

Caspase-8 cleavage was totally abolished in A549 cells transiently transfected with PPARγ dominant negative vector further emphasising the previous observation (Fig. 28).

4.2.8 IL-4 apoptosis involves death domain receptor pathway

Death domain receptors are family of cell receptors which regulate the survival of the cell in response to various factors such as Fas ligand, TNF-α and TRAIL. These receptors upon activation utilise special adapter proteins to activate the caspase cascades. The involvement of death domain receptors in IL-4 and PPARγ induced apoptosis in A549 cells was verified using a dominant negative vector of FADD, a vital adapter protein in the signaling cascade. This mutant lacks the death effector domain, thereby is unable to transmit the signal.

Figure 28 IL-4 induced cleavage of caspase-8 involved death domain receptors.

Cells were transiently transfected with the dominant negative vectors of either PPARg or FADD. The IL-4 stimulated cell lysates were analysed by Western blotting for the cleavage of caspase-8.

As shown in Fig. 28, IL-4 induced cleavage of caspase-8 was completely abolished in cells transfected with FADD dominant negative plasmid, demonstrating the involvement of death domain receptors in IL-4 and PPARγ induced apoptosis in A549 cells.

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4.2.9  Bid cleavage was not induced by caspase-8

The activated caspase-8 can stimulate apoptosis via either direct cleavage and activation of caspase-3 or the mitochondrial route involving the cleavage of Bid and release of cytochrome c into the cytoplasm.

Figure 29 Cleavage of Bid does not occur during IL-4 induced apoptosis.

No cleavage of Bid was observed in IL-4 treated A549 cells indicating the absence of the mitochondrial pathway. Jurkat cells treated for 4 h with 25 µM etoposide were used as positive control.

IL-4, 15-HETE and 15-PGJ2 stimulation failed to induce cleavage of Bid as analysed by western blotting (Fig. 29). This suggests that the caspase-8 directly activates caspase-3 upon IL-4 treatment. As described for type-I cells undergoing apoptosis (Scaffidi et al., 1998).

4.2.10 IL-4 treatment causes activation of Bax and downregulates Bcl-XL

Bcl-XL is an antiapoptotic member of the Bcl-2 family. The anti-apoptotic members of the Bcl-2 family of proteins reside in the outer membrane of the mitochondria and prevent the release of cytochrome c. In the cytoplasm, cytochrome c binds to Apaf1 leading via caspase-9 to the activation of caspase-3.

Figure 30 IL-4 causes downregulation of Bcl-XL in A549 cells.
Incubation of A549 cells with IL-4 causes downregulation of Bcl-XL protein, which was reversed by the treatment of cells with NDGA or by the transient transfection of PPARγ dominant negative vector.

Cells treated with IL-4 showed a marked decrease in the level of Bcl-XL which was completely reversed by the pre-treatment of the cells with NDGA (Fig. 30) and also by the [page 61↓]transient transfection of PPARγ dominant negative vector. The effect of IL-4 and PPARγ ligands on other members of the Bcl-2 family was investigated. In untreated cells, Bax, a pro-apoptotic member of the Bcl-2 family, was present mostly in the cytoplasm and upon IL-4 treatment a large amount was translocated to the mitochondria (Fig. 31). Porin VDAC, a mitochondrial membrane protein was used as a control to show the purity of the mitochondrial and cytoplasmic preparations.

Figure 31 Translocation of Bax to mitochondria.

Translocation of Bax to mitochondria from the cytoplasm was observed upon IL-4 treatment of A549 cells. However, no cytochrome c release was observed from the mitochondria upon IL-4 stimulation. Porin VDAC was used as a control to check the purity of the mitochondrial preparations.

The decrease in the Bcl-XL levels with the concomitant activation of Bax indicates the involvement of the mitochondria in the apoptosis. Regulation by Bcl-2 family of proteins of the efflux of cytochrome c occurs by increasing membrane permeability and alteration of the inner mitochondrial membrane potential. The released cytochrome c interacts with Apaf-1 in the cytoplasm and eventually causes the activation of caspase-3. However, no cytochrome c release was observed upon IL-4 treatment. Thisimplies a cytochrome c-independent functional interplay betweenthe pro- and anti-apoptotic members of the Bcl-2 family in IL-4-induced apoptosisin A549 cells.

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4.3  12/15-Lipoxygenase exhibits hepoxilin synthase activity

4.3.1 Rinm5F cells express hepoxilin synthase activity

Cultured Rinm5F cells were incubated with AA for 20 minutes and the products were analysed as ADAM ester derivatives by HPLC monitoring fluorescence detection. As shown in Fig. 32, a HXA3 peak was observed, which co-migrated with an authentic standard.

Upon longer incubation, e.g. 40 min., HXA3 peak was substantially reduced and a more polar peak appeared (not shown), which has earlier been characterised as the hydrolysis product 8(S/R),11(R),12(S)-trihydroxy-eicosa-5Z,9E,14Z-trienoic acid and 8(S/R),9(R),12(R)-trihydroxy-eicosa-5Z,8Z,14Z-trienoic acid, trivially known as trioxilins A3 (TrXA3). As these hepoxilins are hydrolysed by cellular epoxide hydrolases, HXA3 formation was determined in Rinm5F cells pretreated with 100 µg/ml trichloro-propylene oxide (TCPO), an inhibitor of epoxide hydrolases. After 40 minutes incubation with AA a significant increase in HXA3 formation was observed (Fig. 33) without appearance of a polar peak.

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Figure 33 TCPO treatment increases the formation of HXA3.

Rin cells were incubated with AA, lipds were extracted derivatised and separated on HPLC. TCPO, an epoxide synthase inhibitor inhibits the formation of trioxilins, thereby increasing the amount of HXA3 detected.

To confirm the chemical structure of HXA3 from Rinm5F cells, the incubation mixture was extracted, the residue acid-hydrolysed (to transform HXA3 to stable trioxilins) and converted to methyl-silyl derivatives for GC-MS analysis. As shown in Fig. 34B (panel II), a single peak characterising trioxilin A3 (TrXA3) (Fig. 34A) was observed. No TrXB3 was detected, indicating the absence of HXB3 formation. Heat-denatured (90oC for 10 min.) cell lysate failed to produce any HXA3 from 12-HpETE and no 12-HETE from AA, suggesting an enzymatic pathway of formation (Fig. 34B, bottom panel) and the presence of 12-LOX activity was essential in this process.

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Figure 34 Formation of HXA3 in Rin cells.

Rin cells were incubated with 12-HpETE and the extracted lipids were derivatised, separated and identified by GCMS.

4.3.2 Glutathione peroxidases inhibit the formation of HXA3

Recently, the prominent role of PHGPx as a 12(S)-HPETE reductase has been demonstrated in human platelets (Sutherland et al., 2001). In absence of cGPx the reduction of 12(S)-HPETE to 12(S)-HETE was taken over by PHGPx. But, inactivation of both selenoenzymes cGPx and PHGPx by the depletion of glutathione, led to accumulation of 12(S)-HPETE, the metabolism of which was consequently diverted to HXA3 and HXB3 formation. Since Rinm5F cells are almost devoid of cGPx as well as PHGPx (Lortz et al., 2000), the formation of hepoxilin was predicted.

Figure 35 Formation of HXA3 was regulated by cGPx.

Representative mass chromatogram of normal Rin and Rin stably transfected with cGPx and AA. Transfected cells show no production of TrXA3.

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As expected these cells synthesised hepoxilin when incubated with AA as substrate (Fig. 35, top panel). Conversely, the presence of GPx should diminish the formation of HXA3. Indeed, upon incubation with AA cultured RINm5F cells, stably transfected with cGPx did not produce any HXA3 as determined by GC-MS (Fig. 35, bottom panel). Incubation with 12(S)-HpETE of native or cGPx-transfected RINm5F cells reproduced identical results, to those obtained with AA. The chemical characterisation of the hepoxilin by HPLC and GCMS showed that only HXA3 and no HXB3 was formed, which another substantiation of the enzymatic nature of HXA3 synthesis in Rinm5F cells.

4.3.3 Rat 12/15-LOX exhibits intrinsic HXA3 synthase activity

Lipohydroperoxidase activity has been attributed to 12/15-Lipoxygenases (Veldink et al., 1997). This activity suggests a role for 12/15-LOX in the synthesis of HXA3. HeLa cells do not exhibit any intrinsic 12-LOX activity, so these cells were utilised for transfection with rat 12/15-LOX. Upon incubation of cultured transfected cells with AA, neither 12(S)-HETE nor HXA3 was produced, suggesting the complete down-regulation of 12-LOX by selenoenzymes present in the cell (Fig. 36, top panel). However, pretreatment of transfected cells with by 2 mM diethyl maleate (DEM), which depletes cellular glutathione (GSH) and thus inhibits glutathione peroxidases (Imai et al., 1998), caused significant enhancement of HXA3 synthesis (Fig. 36, bottom panel). As no HXB3 formation was detected in GSH- and GPx-depleted cells, the specific HXA3 synthase activity of 12/15-LOX can be observed.

Figure 36 Rat 12/15-LOX exhibits hepoxilin synthase activity.

HeLa cells were transfected with 12/15-LOX plasmid and reacted with AA.

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4.3.4  Hepoxilin synthase activity in Rin cells was depleted by a 12/15-LOX antibody

As seen in the previous experiment, upon 12/15-LOX transfection of HeLa cells hepoxilin synthase activity was observed. To confirm these findings, Rin cell lysates were subjected to immunoprecipitation with 12/15-LOX specific antibody. The lysate, immunoprecipitate and the 12/15-LOX depleted lysate were incubated with 12-HpETE and analysed by GCMS for the production of TrXA3.

Figure 37 12/15-LOX antibody depletes hepoxilin synthase activity from RIN cells.

Rin cell lysate was subjected to immunprecipitation with 12/15-LOX antibody. The total lysate, immunoprecipitate and the depleted lysate were incubated with 12-HpETE and the production of TrXA3 was measured by GCMS. Similarly lipoxygenase activity was determined by incubating the lysates with AA and analysing with HPLC. 13-HODE was used as internal standard for quantification of both TrXA3 and 12-HETE. (n = 3).

The immunoprecipitated protein shows high amount of hepoxilin synthase activity (fig. 37). There was a significant reduction in the hepoxilin synthase activity observed in the Rin cell lysate after immunoprecipitaion. Since the amount of protein was higher than the amount of antibody complete depletion was not observed. Simultaneously the lipoxygenase activity was also determined by the incubation of lysates with AA and measuring the formation of 12-HETE produced by HPLC. The depletion in the lipoxygenase activity paralleled the hepoxilin synthase activity. 13-HODE was used as an internal standard to normalise the extraction process and for quantification. Nevertheless, these observations confirm the hypothesis that 12/15-LOX was responsible for the hepoxilin production.

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4.3.5  Recombinant 12/15-LOX protein exhibits hepoxilin synthase activity

The full length 12/15-LOX gene was amplified from mRNA prepared from RIN cells. The PCR product was cloned into pET15b bacterial expression vector. E.coli BL21 DE3 cells were transformed with this vector and the recombinant protein synthesis was induced by 1 mM IPTG for 3 hours.

Figure 38 Recombinant 12/15-LOX exhibits hepoxilin synthase activity.

Recombinant 12/15-LOX was expressed in bacteria. The bacterial lysate was reacted with 12-HpETE. The extracted lipids were derivatised with ADAM reagent and separated by HPLC. The recombinant enzyme produces a clear HXA3 peak.

The cell lysate was incubated with 12-HpETE and the extracted lipids were subjected to HPLC. A distinct HXA3 peak was observed (fig. 38).This experiment confirms the hepoxilin synthase activity of 12/15-LOX.

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4.3.6  12/15-LOX produces epoxyhydroxy compounds with 15(S)-HpETE

To test whether the lipohydroperoxidase activity of 12/15-LOX was applicable to other substrates, 15(S)-HpETE was reacted with the recombinant enzyme. The GCMS chromatogram shows the production of two compounds which were identified as 11,12,15-trihydroxyeicosatrienoic acid and 11,14,15-trihydroxyeicotrienoic acid (THETA) (fig. 39). Identical compounds were identified in reaction of rabbit 15-LOX and AA (Pfister et al., 1998). Furthermore, 15-LOX did not produce any epoxyhydroxy compounds with 12-HpETE. Thus, rabbit 15-LOX appears to use only 15-HpETE as substrate while rat 12/15-LOX can accommodate a larger variety of substrates. This unexpected substrate specificity could be due the larger predicted active site volume in the 12/15-LOX as compared to the rabbit 15-LOX.

Figure 39 Rat 12/15-LOX produces epoxyhydroxy compounds from substrates other than 12-HpETE.

Panel A: Representative mass chromatogram (m/z 243) for the reaction between recombinant rabbit 15-LOX and 12-HpETE. No TrXA3 was detected. Panel B: Chromatogram (m/z 173) of 15-LOX and AA. Panel C: Chromatogram (m/z 173) of rat 12/15-LOX with 15-HpETE. In panel B and C formation of compound I and II were observed. The fragmentation profile of the two compounds allowed their identification as I : 11,14,15-THETA and II: 11,12,15-THETA.

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Figure 40 Positional determinant of 12-Lipoxygenation reaction is important for the hepoxilin synthase activity.

Recombinant rat 12/15-LOX, wild type rabbit 15-LOX and I418A mutant of 15-LOX were incubated with 12-HpETE and the products were analysed by GCMS. TrXA3 formation was quantified by comparison to the internal standard (15-HETE). (n = 3).

The mutation of isoleucine 418 to alanine (I418A) in rabbit 15-LOX was observed to shift the positional specificity of this towards 12-lipoxygenation (Sloane et al., 1991). Formation of TrXA3 was measured with recombinant rat 12/15-LOX, wild type rabbit 15-LOX and I418A mutant of 15-LOX. As observed before (fig. 39) wild type 15-LOX did not produce any TrXA3 with 12-HpETE, however, the I418A mutant showed a substantial increase in the formation of TrXA3 (fig. 40). This demonstrates the importance of the positional specificity of the 12-lipoxygenase reaction for the hepoxilin synthase activity. These data attest the hypothesis that 12/15-LOX possess a hepoxilin synthase activity and can accommodate different substrates like 12-HpETE and 15-HpETE.

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