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5  Discussion

5.1 Upregulation of 15-LOX-1 expression by IL-4 in lung epithelial cells

Acetylation of proteins is a common principle to modify their biological activity. It impacts protein chemical properties and, thus may alter protein-protein interaction, DNA recognition and protein stability. Histones were the first proteins that have been identified as targets for protein acetylation. Although there are several lines of experimental evidence suggesting the importance of histone acetylation in the transcription of a variety of genes (Grunstein, 1997), its precise role in nucleosome remodelling is still elusive. Several families of histone acetyltransferases (PCAF/GCN5, CBP/p300, TAF(II)250, SRC-1, MOZ) have been characterized in the past and, recently even non-histone nucleic acid binding proteins, such as HMG-1, p53, GATA1 have been identified as acetylation substrates (Sterner et al., 1979; Gu and Roeder, 1997). The consequence of acetylation of these regulatory proteins depends on the internal sites of acetylation. For instance, HMG-1 is acetylated at its DNA binding site, which results in the disruption of its DNA-binding capabilities (Sterner et al., 1979). In contrast, other transcription factors such as p53, GATA1, E2F1 are acetylated outside their DNA binding site and this results in stimulation of DNA-binding (Gu and Roeder, 1997). Sequence alignments have indicated that STAT6 contains several potential acetylation sites and its acetylation has already been reported before (McDonald and Reich, 1999). However, this process was independent of IL-4. The data presented in this study clearly indicate that IL-4 induced transcription of the 15-LOX-1 gene requires upregulation of STAT6 acetylation, which is mainly due to activation of the acetyltransferase activity of CBP/p300. It should, however, be stressed that the acetylation degree of cellular proteins is a resultant of acetylating and deacetylating processes. Thus, an increase in the acetylation degree of STAT6 can either be achieved by activation of acetyltranferases (CBP/p300) and/or by inhibition of deacetylases. Deacetylases have been shown to occur in the nucleus and appear to play an important role in transcriptional repression (Downes et al., 2000). Whether they are recruited by nuclear hormone receptors bound to certain nuclear corepressors is not clear (Ordentlich et al., 2000). It was found that sodium butyrate (non-specific inhibitor of cellular HDAC) alone is capable of inducing 15-LOX-1 expression in A549 cells suggest that transcriptional repression of the 15-LOX gene in resting cells may be due to a preponderance of deacetylating processes over acetyltransferases. It would be of particular interest to elucidate whether it is a general principle for transcriptional repression of 15-LOX-1 gene in mammalian cells. Recently, Kamitani et al., 2001, have observed that treatment of colorectal cell line, Caco-2 with sodium butyrate and other histone deacetylase inhibitors causes an [page 72↓]upregulation of 15 LOX-1 expression and found that this upregulation is linked to the degree of histone acetylation. Recently, the expression of IL-8 gene by lung epithelial cells upon challenge by particulate matter was also shown to be upregulated by histone acetylation (Gilmour et al., 2003). Moreover, the question of whether similar mechanisms may be involved in transcriptional activation of other IL-4-inducible genes remains to be investigated in the future.

In A549 cells the expression of the 15-LOX-1 gene may be inhibited under resting conditions because non-acetylated histones formed by HDAC are bound to the 15 LOX-1 promoter. This mode of transcriptional repression has been reported for a variety of inducible genes and appears to be well characterised (Grunstein, 1997). Histone binding to genomic DNA forms a condensed nucleosomal structure and there is no possibility for the binding of specific transcription factors. Acetylation may induce conformational changes of the histone octamer, which then may provide access to transcription factors for binding to the promoter of relevant genes. Originally, it was postulated that phosphorylation of STAT6 would be sufficient to allow its binding to the 15-LOX-1 promoter. However, the present data indicates that this may not be true. In A549 cells IL-4 induces STAT6 acteylation in addition to phosphorylation and both reactions appear to be required for translational activation of the 15-LOX-1 gene. This conclusion may be drawn form the following experimental data: i) IL-4 increases the activity of cellular acetyltransferases (Fig. 9), particularly of CBP/p300, which are capable of acetylating STAT6 (Fig. 17). ii). The viral oncoprotein wtE1A, an inhibitor of acetyltransferase activity of CBP/p300, prevented STAT6 acetylation, and expression of the functional enzyme. In contrast, its non-inhibitory mutant E1AmCBP was unable to do so (Fig. 17,18,19). iii) Inhibition of STAT6 acetylation by wtE1A prevented STAT6 binding to the 15-LOX-1 promoter (Fig. 18). In contrast, the E1AmCBP mutant did not inhibit the CBP/p300 acetylase activity and also did not prevent STAT6 binding. iv) The histone deacetylase inhibitor sodium butyrate synergistically induced the IL-4-stimulated 15-LOX-1 expression.

Since further acetylation of STAT6 by CBP/p300 acetyltransferases takes place inside the nucleus, tyrosine phosphorylated STAT6, which is inevitably required for homodimerization and subsequent nuclear translocation, becomes an essential precursor. This is strongly supported by the inhibition of STAT6 acetylation by genistein (Fig. 11 and 12). No effect of wtE1A oncoprotein on the tyrosine phosphorylation of STAT6 was observed as checked with anti-phospho-STAT6 antibody in Western blot.

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Phosphorylation of transcription factors is a rapid process and our in vitro binding assays indicated that phosphorylated STAT6 quickly binds to the naked 15-LOX-1 promoter. On the other hand, in vivo expression of the 15-LOX-1 mRNA in intact A459 cells requires at least 11 h and similar observations have been reported for other cytokines (Roy and Cathcart, 1998). This obvious discrepancy together with the results presented in Fig. 12 indicated that in vivo the binding of phosphorylated STAT6 to the 15-LOX-1 promoter appears to be inhibited during the first 11 h of IL-4 treatment. Although the detailed mechanism of the inhibitory processes is still unclear our data suggest that the binding of non-acteylated histones may be involved. Acetylation of histones, which appears to be a delayed process in A549 cells, may overcome this inhibitory process, so that acetylated STAT6 can bind to the 15-LOX-1 promoter. For the time being, the reasons for the delayed acetylation of histones and STAT6 remain obscure. Although the cellular acetyltransferase activity is upregulated within 1 h following IL-4 exposure, acetylated histones and acetylated STAT6 could only be detected after a 9 h incubation period.

In summary, it can be concluded from the data that the mechanism of transcriptional activation of the 15-LOX-1 gene by IL-4 does not follow the conventional activation pathway of IL-4 inducible genes. Acetylation of both histones H3 and STAT6 is essentially required for transcriptional activation of the 15-LOX-1 gene, and this acetylation is mainly due to the acetyltransferases activity of CBP/p300. These observations suggest that acetylation is an additional step required in the normal signal transduction pathway induced by IL-4 in these cells. The implication of acetylation in other IL-4 induced pathways, however, needs to be confirmed.

5.2 Induction of apoptosis by 15-LOX-1

In the present study, it has been shown that 15(S)-HETE is bound as a ligand to PPARγ transcription factors (Fig. 24) and is an effector of apoptosis (Figs.20-23). Moreover, treatment of cells with NDGA, a 15-lipoxygenase inhibitor, prevented the PPARγ activation and apoptosis. Identical results were also obtained with PPARγ ligand 15-PGJ2. To further substantiate the crucial role of 15(S)-HETE for apoptosis via PPARγ transcription factor, dominant negative form of PPARγ was used, in which two amino acids (L468A and E471A) have been mutated, thus impairing transcriptional activation and co-factor recruitment (Adams et al., 1997). Transfection of PPARγ dominant negative in A549 cells strongly inhibited the IL-4-induced and 15-PGJ2-induced apoptosis (Fig. 26), supporting the prominent [page 74↓]role of 15(S)-HETE and 15-PGJ2 as effectors of apoptosis via PPARγ pathway. It must be mentioned that the concentrations of 15-HETE (30 µM) and 15-PGJ2 (5 µM) used were far higher than the physiological concentrations in the cell. However, this difference should not totally discredit the role of the compounds in the apoptotic process as inside the cell the local concentrations could attain such values. Experiments with normal bronchial epithelial cells BEAS-2B (fig. 23) confirmed the observations in A549 cells and thus underlined the importance of these observations in human allergic inflammatory reactions.

15-LOX-1 is a lipid peroxidising enzyme and has the capability of damaging lipid membranes especially the organelle membranes as seen during the maturation of erythrocytes. This entails an important role for this enzyme in the process of cell death either through apoptosis or necrosis. However, not all LOX expressing cells undergo apoptosis and there is apoptosis in the absence of 12/15-LOX. One of the factors with an regulatory role may be PHGPx. The ability of PHGPx to scavenge the lipid hydroperoxides may play an important role in limiting the damage caused by lipoxygenase. PHGPx has been shown to play an important role in inhibiting free radical induced damage of the mitochondria and consequently apoptosis (Imai et al., 1996; Nomura et al., 1999). IL-4 treatment of A549 cells causes a simultaneous increase in the lipoxygenase activity and a decrease in the PHGPx activity (Schnurr et al., 1996). This leads to a condition of high oxidative stress and may result in cell death. The reduction of the defensive capability of PHGPx could be the decisive step. However, there was no cytochrome c release observed upon IL-4 stimulation (fig. 31) indicating a lack of mitchondrial membrane leakage and damage. Cytochrome c release is a very rapid process, often occurring within the first 30 minutes of the apoptotic process (Lum et al., 2003). Thus the release of cytochrome c may have been finished by the time the assay was performed. The lipoxygenases constitute only one of a number of pathways operative in the death of a cell. A number of other factors, perhaps activated by PPARγ could play a role in the apoptosis observed. Thus, the importance of 15-LOX in the apoptotic process needs to be investigated in detail.

The exact downstream processes of PPARγ activation are still unclear. Involvement of death domain receptor in IL-4-induced apoptosis has been observed. The application of FADD dominant negative vector (Hofmann et al., 2001), lacking the death effector domain, abrogated the apoptotic signal induced by IL-4 or PPARγ (Fig. 28) . The involvement of death domain receptors in IL-4-induced apoptosis can be observed by the cleavage of caspase-8 to active subunits p41/42 and p18 (Fig. 27,28) This cleavage and activation is inhibited by [page 75↓]NDGA and PPARγ dominant negative vector demonstrating the vital importance of 15-LOX and PPARγ in the activation of apoptosis signal in this cellular system. Activated caspase-8 has been proposed to stimulate apoptosis through two parallel pathways (Scaffidi et al., 1998). In type-I cells, capsase-8 directly cleaves and activates caspase-3. Type-II cells utilize the mitochondrial pathway through the cleavage of Bid and subsequent release of cytochrome c to amplify the apoptotic signal. Caspase-3, an effector caspase further cleaves PARP and other cellular proteins to cause apoptosis. The IL-4 treated cells do not exhibit Bid activation (Fig. 29), thus suggesting the involvement of the type-I pathway. Earlier, it has been shown that PPARγ promotes the TRAIL induced apoptosis (Ji et al., 2001). TRAIL utilizes various types of death receptors like DR3, DR4 and DR5 to trigger apoptosis (Baker and Reddy, 1998; Ashkenazi and Dixit, 1998). However, it is intriguing to note that IL-4-induced apoptosis in A549 is mediated simultaneously through two different pathways, i.e. through direct activation of caspase-3 and through mitochondrial pathway involving Bax. The activation of Bax and its subsequent translocation to the mitochondria along with the decrease in Bcl-XL can account for the mitochondrial pathway, however, no cytochrome c release was observed. A number of factors other than cytochrome c, when released from the mitochondria can activate apoptosis (Garl and Rudin, 1998; Matsuyama and Reed, 2000) Thus, the scenario can be explained in the following way: in type I cell death, binding of 15(S)-HETE to PPARγ transcription factor leads to generation of active caspase-8 through activation of FADD protein (Fig 27,28), which subsequently activates downstream effector caspase-3. In IL-4-stimulated cells the binding of 15(S)-HETE to PPARγ transcription factor downregulates Bcl-XL (Fig. 30,31) and the activation of Bax commits the cell to apoptosis via the intrinsic pathway. Bax has also been shown to be involved in a number of apoptotic pathways, especially the DNA damage-induced apoptosis involving p53 (Wu and Deng, 2002) without participation of death receptors. Antidiabetic thiazolidinediones, potent PPARγ agonists, have been observed to induce apoptosis in vascular smooth muscle cells through p53 and Gadd45 pathway, although it is not clear whether PPARγ itself is the effector (Okura et al., 2000). In non small cell lung cancer cells, it has been shown that troglitazone induced DNA damage-inducible Gadd 153 gene. Bax and p53 form an important pathway in DNA damage induced apoptosis (Satoh et al., 2002). Caspase-3 has also been observed to activate the intrinsic apoptotic pathway by the cleavage of anti-apoptotic Bcl-xL and Bcl-2 to pro-apoptotic components (Cheng et al., 1997; Clem et al., 1998). Thus, caspase-3 activated by other [page 76↓]pathways can activate the mitochondrial route and provide a positive feedback loop for caspase-3 production leading to apoptosis.

In asthma, an upregulation of IL-4 secretion in the blood and higher levels of 15-HETE in the lung and bronchial tissue have been found. It is, therefore, hypothesised that IL-4-induced apoptosis is one of the major causes responsible for the hypertrophy of the bronchial smooth muscle, denuded surface epithelium, thickened basement membrane and infiltration of eosinophils, lymphocytes and mononuclear phagocytes as well as for the apoptotic lesions observed in the lung tissue of asthma patients. It has been reported that PPARγ ligands induce apoptosis in lung cancer cells, and this may be beneficial for the therapy of such cancers (Theocharis et al., 2002; satoh et al., 2002; Inoue et al., 2001; Tsubouchi et al., 2000). In contrast, in chronic inflammatory diseases, such as COPD, the loss of alveolar structures in the lung tissue due to apoptosis may worsen the lung function (Kasahara et al., 2000).

5.3 Biosynthesis of HXA3 and its regulation by glutathione peroxidases

Rinm5F rat insulinoma cells, which possess leukocyte-type 12-LOX, are devoid of cGPx and PHGPx (Lortz et al., 2000),produced only HXA3 when incubated with AA or 12(S)-HpETE (Fig. 32,33). Moreover, no HXA3 was detected in the lysate from heat-inactivated cells (Fig. 34). But, cultured cells stably transfected with cGPx or PHGPx-cDNA did not show any formation of HXA3 (Fig. 35). These results implied the presence of HXA3 synthase-like activity in Rinm5F cells and its regulation by glutathione peroxidases. It has been earlier reported that 12/15-LOXs exhibit lipohydroperoxide activity and produce epoxyhydroxy acids from AA (Veldinck et al., 1997; Kühn, 1996; Kühn et al., 1986a). These reports along with other reports on the mechanism of formation of hepoxilin suggested that rat 12/15-LOX could be responsible for the hepoxilin synthase activity. To test the hypothesis recombinant rat 12/15-LOX was overexpressed in bacteria. This enzyme, upon incubation with 12-HpETE or AA, produced HXA3 (fig. 38). Further confirmation was obtained by performing depletion experiments with 12/15-LOX specific antibodies, which suggest the cohabitation of hepoxilin synthase and lipoxygenase activity in the same protein. Immunoprecipitation with the specific antibody depleted the hepoxilin synthase activity in Rin cell lysates (fig. 37) along with the LOX activity and the activity was recorded in the immunoprecipitated protein. It must be mentioned at this point that immunoprecipitation experiments did not completely deplete the enzyme activity in the lysates. It was of interest to test whether the lipohydroperoxidase activity could be verified using other substrates. 15-HpETE offered similar structural features to test this possibility. Recombinant 12/15-LOX was observed to form two trihydroxy acid [page 77↓](acid hydrolysed from epoxyhydroxy acids) from 15-HpETE. An earlier work (Pfister et al., 1998) also reported the formation of two trihydroxy acids from AA using rabbit aortic protein extracts. These were identified from their fragmentation patterns as 11,12,15 trihydroxyeicosatrienoic acid (THETA) and 11,14,15-THETA (fig. 39). The fragmentation pattern in the case of 12/15-LOX confirmed that the two THETAs were observed with 15-HpETE as substrate. According to the chemistry of the lipohydroperoxidase reactions, the predicted products of such a reaction with 15-HpETE would have been the THETAs. Further, recombinant rabbit 15-LOX also produced these compounds with AA. However, when 12-HpETE was used as substrate with rabbit 15-LOX, no hepoxilin synthesis was observed. This suggests that the rat 12/15-LOX has a broader range of substrates as compared to the rabbit enzyme. Such a phenomenon could be due the slightly larger predicted volume of the rat 12/15-LOX active site, which would allow the accommodation of different substrates. Furthermore, the shifting of the positional specificity of rabbit 15-LOX to 12-lipoxygenation by I418A mutation caused the formation of hepoxilin (fig. 40). This suggests the importance of the positional determinants of 12-lipoxygenase reaction for the hepoxilin synthase activity. The lipohydroperoxidase activity offers an interesting facet to the enzymatic nature of the lipoxygenases.

HeLa cells overexpressing rat 12/15-LOX did not produce any HXA3 and 12(S)-HETE under normal conditions due to the presence of abundant glutathione peroxidases. There was, however, a drastic increase in the HXA3 formation upon treatment with DEM, which depletes cellular GSH and inhibits PHGPx thus, elevating the overall hydroperoxide tone. These data also confirm the hypothesis that the rat 12/15-LOX exhibits an intrinsic HXA3 synthase activity. The activity was found to be finely regulated by cellular glutathione peroxidases cGPx and PHGPx. This regulation by glutathione peroxidases is in line with earlier observations reported for 5-, 12- and 15-LOX (Weitzel and Wendel, 1993; Sutherland et al., 2001; Schnurr et al., 1996). Normally, in various cell types the presence of glutathione peroxidases exerts the primacy of the reduction pathway over the hepoxilin pathway. However, in experiments with HeLa cells overexpressing the rat 12/15-LOX seemingly selenoenzymes diminished the cellular peroxide tone to such an extent that even a minimum peroxide level essential to trigger the activation of 12-LOX may not be present.

On the basis of these data, it should however be stressed that the mechanism underlying the activation of HXA3 synthase activity could be primarily due to competition for the same substrate 12-HpETE. Formation of HXA3 implies the removal of hydroperoxides and thus can be regarded as a counteraction to the permanent oxidative-stressed cellular status. Thus, [page 78↓]synthesis of hepoxilins plays an important role in the processing of cellular hydroperoxides and hence in the overall regulation of the 12-LOX pathway in cells deficient in antioxidant enzymes.

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