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Transcription Factor1
Eicosanoid and Lipid Research Division, Department of Gynecology, University Medical Center Benjamin Franklin, Free University Berlin, Berlin, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(PPAR) nuclear
receptors in macrophages and A549 cells. In this study we demonstrate
that 15(S)-HETE binds to PPAR nuclear receptors and induces
apoptosis in A549 cells. Moreover, pretreatment of cells with
nordihydroguaiaretic acid, a 15-lipoxygenase inhibitor, prevented
PPAR activation and apoptosis. The latter was accomplished by the
interaction of the 15(S)-HETE/PPAR complex with the adapter protein
Fas-associating protein with death domain and caspase-8, as shown by
transfection of Fas-associating protein with death domain dominant
negative vector and cleavage of caspase 8 to active subunits p41/42 and
p18. Whereas IL-4 and PPAR ligands failed to induce cleavage of Bid
and release of cytochrome c from mitochondria, they
caused translocation of the proapoptotic protein Bax from cytoplasm to
mitochondria with a concomitant decrease in the Bcl-xL
level. We therefore believe that in unstimulated cells
Bcl-xL and Bax form a heterodimer, in which
Bcl-xL dominates and prevents the induction of apoptosis,
whereas in IL-4-stimulated cells the 15(S)-HETE/PPAR complex
down-regulates Bcl-xL, and the resulting overweight of Bax
commits the cell to apoptosis via caspase-3. However, this pathway does
not rule out the direct caspase-8-mediated activation of caspase-3. In
conclusion, IL-4-induced apoptosis may contribute to severe loss of
alveolar structures and infiltration of eosinophils, mononuclear
phagocytes, etc., into the lung tissue of chronic asthma
patients. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Metabolites of arachidonic acid play a fundamental role as mediators in the development of airway inflammation in asthmatics (4). Although the pivotal role of 5-lipoxygenase in asthma led to the development of numerous drugs, a real breakthrough in combating asthma has not yet been achieved. Today we know that the bronchial hyper-reactivity is not caused by increased release of mediators of bronchoconstriction and inflammation alone; rather, the increased sensitivity of bronchial receptors toward these mediators is important in the pathogenesis of asthma (2). The lung epithelial cells possess 5- and 15-lipoxygenases as well as cyclooxygenases, which produce a variety of metabolites, such as 5-hydroxyeicosatetraenoic acid (5-HETE),3 15-HETE, and leukotrienes, and prostanoids, respectively (5). The expression and secretion of these enzymes are tightly regulated in different types of cells during airway inflammation (6, 7, 8). Specially, the expression of 15-lipoxygenase (15-LOX) enzyme is significantly increased in human bronchial epithelial cells and monocytes (9, 10). Consequently, large amounts of 15(S)-HETE were found in bronchial epithelial cells of asthmatic and emphysema patients (11). It is also expressed in macrophages of atherosclerotic lesions (12). 15-LOX has been shown to play a key role in the breakdown of mitochondria during erythrocyte maturation, development of fiber cells in eye lens, and actin polymerization during phagocytosis of apoptotic cells (13, 14, 15). Since 15-LOX dioxygenates not only free arachidonic acid, but also biomembrane phospholipids (16), a considerable alteration in the affinity of various receptors with respect to 15(S)-HETE and other lipid mediators may be expected. Thus, exposure of human tracheal epithelial cells to ozone was found to increase 15(S)-HETE production and its subsequent esterification into phospholipids (16).
IL-4, a proinflammatory cytokine, is released in large amounts during
the allergic inflammatory response in asthma and plays a key role in
the development of airway inflammation (17). Therefore,
Abs raised against the IL-4R have been reported to alleviate the
symptoms of asthma, highlighting their potential therapeutic
application (17). Earlier, we and others have shown that
IL-4 up-regulates 15-lipoxygenase in airway epithelial cells
(18), monocytes (9), and A549 lung epithelial
carcinoma cells (19) via the Janus kinase/STAT6 pathway
(20, 21, 22). In A549 cells activation of 15-LOX by IL-4
required the coactivation of histone acetyltransferases CREB-binding
protein/p300 and led to a sizable production of 15(S)-HETE
(21). IL-4 has also been shown to up-regulate the nuclear
receptor and transcription factor PPAR and transcription of the CD36
gene (23, 24). PPAR receptors belong to the family of
PPARs and were initially characterized as regulators of adipocyte
differentiation and lipid metabolism (25, 26, 27, 28). A number of
lipids, including 15(S)-HETE, have been shown to be their ligands in
adipocytes and various other tissues (26, 27).
Pathogenesis of asthma is also related to the balance between survival
and apoptosis of inflammatory cells. A high degree of apoptosis has
been reported in the epithelium of asthma patients (3, 29), which is enhanced by inhaled as well as oral
corticosteroids (3). In the present study we have shown
for the first time that the 15(S)-HETE produced by IL-4-activated A549
cells physically binds to PPAR transcription factor as a ligand and
leads to apoptosis of A549 cells via the death receptor and caspase-3
pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 549 cells (lung adenocarcinoma cells) were cultivated in DMEM containing 10% FCS, 50 U/ml of penicillin, and 50 µg/ml of streptomycin. For microscopy, cells were grown on glass coverslips, treated for 72 h with 670 pM (10 ng/ml) IL-4 (R&D Systems, Wiesbaden-Nordenstadt, Germany), and then assayed for apoptosis. Nordihydroguaiaretic acid (NDGA; (Sigma-Aldrich, Deisenhofen, Germany) was used as a lipoxygenase inhibitor at a concentration of 10 µM. Normal human bronchial epithelial cells (BEAS-2B) were obtained from American Type Culture Collection (Wesel, Germany) and were cultivated in a modified LHC-9 medium with all the necessary growth factors (supplied by Clonetics, Palo Alto, CA).
Assays for apoptosis
Apoptosis was detected by staining cells with annexin V and propidium iodide and by TUNEL assay. For the annexin V assay, coverslips were washed with PBS and incubated for 15 min at room temperature with a solution of annexin V fluos and propidium iodide (Roche, Mannheim, Germany). Cells were then washed twice with PBS and observed under a fluorescence microscope. Normal cells do not stain, while apoptotic cells are stain green (annexin V fluos), and necrotic cells stain red (propidium iodide). A minimum of 200 cells were counted.
TUNEL assay was performed by washing paraformaldehyde-fixed cells on a coverslip once with PBS and then were permeabilized using 0.5% saponin at room temperature for 30 min. After washing with TdT buffer, cells were incubated with 0.5 µM biotin dUTP and 150 U/ml of TdT in 30 µl of TdT buffer (Roche) in a humidified chamber at 37°C for 30 min. After washing twice with PBS, the cells were incubated with a 1/1000 solution in PBS of streptavidin-conjugated HRP (Life Technologies, Karlsruhe, Germany) for 10 min at room temperature. Coverslips were then washed for 30 min with three washes of PBS. Color was developed with True Blue (KPL Laboratories, Wedel, Germany) peroxidase substrate, and coverslips were observed under a light microscope. Apoptotic cells were stained blue.
A cell death detection ELISA kit (Roche) was used according to the manufacturer’s instructions to quantify apoptosis in cells. Cells (104) were plated on 96-well plates in DMEM. IL-4 (670 pM) or other effectors of apoptosis with or without inhibitors were added, and apoptotic cell death was determined after 72 h with the above-mentioned ELISA kit.
Caspase-3 assay
The caspase-3 assay was performed using acetyl-DEVD-para-nitroaniline (Ac-DEVD-pNA) as substrate. Briefly, cells were lysed with lysis buffer (50 mM HEPES (pH 7.4), 100 mM NaCl, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 1 mM DTT, and 100 µM EDTA) and centrifuged at 10,000 x g for 10 min. Protein (100 µg) was incubated with a 200-µM solution of Ac-DEVD-pNA (Calbiochem, Schwalbach, Germany) in reaction buffer (lysis buffer with 10 mM DTT and 10% glycerol) at 37°C. The development of yellow color at 405 nm indicated caspase-3 activity. The reaction was monitored periodically for 3–4 h. The rate of reaction was calculated as the difference in absorbance at 405 nm per unit time. The results were represented as the fold increase in caspase-3 activity over the control reactions. Z-Val-Ala-Asp(OMe)-CH2F (Z-VAD-FMK; Calbiochem, Germany) was used as an inhibitor of caspase-3 at a concentration of 100 µM 1 h before standard treatment.
Western blot analysis
Cells were scraped in ice-cold RIPA buffer (PBS, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate containing 1 mM PMSF, 1 mg/ml pepstatin, and 1 mg/ml leupeptin). Cell homogenates were collected by centrifugation at 12,000 rpm at 4°C. Protein concentrations were determined using Lowry’s assay (Bio-Rad, Munich, Germany). SDS-PAGE electrophoresis was performed with 50 µg of each protein on polyacrylamide gels of varying concentrations. The protein was transferred onto a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) by a semidry transfer method. After blocking with 5% skimmed milk solution in PBS and 0.05% Tween 20, the blot was incubated with the primary Ab for 1 h. A 5-min wash was followed by incubation with HRP-conjugated secondary Ab (Santa Cruz Biotechnology, Heidelberg, Germany) for 1 h. The signal was visualized using chemiluminescent substrate (Santa Cruz Biotechnology). The primary Abs used were anti-Bcl-xL (ICN, Eschwege, Germany), anti-caspase-3 (Santa Cruz Biotechnology), anti-caspase-8 and anti-Bid (Cell Signaling Technologies, Beverly, MA).
Plasmids and transfection
For PPAR reporter assay, well-characterized PPAR-responsive
promoter region for acyl-coenzyme A oxidase 581–471(581–471) fused to the
minimal globin promoter upstream of a luciferase reporter (pGL3 Basic;
Promega, Mannheim, Germany), termed pPPAR-LUC, was used.
Cotransfections were performed with Rous sarcoma virus
promoter-galactosidase to normalize the transfection efficiency.
Transient transfections with various plasmids were performed with
Polyfect transfection reagent (Qiagen, Hilden, Germany) and 1.5
µg of DNA. PPAR dominant negative (PPAR-DN) plasmid was a gift
from Prof. V. K. K. Chatterjee (Oxford University, Oxford,
U.K.), and Fas-associating protein with death domain double negative
(FADD-DN) plasmid was a gift from Dr. M. L. Schmitz (German Cancer
Center, Heidelberg, Germany). Luciferase and galactosidase activities
were measured according to the manufacturer’s (Promega)
instructions.
Detection of PPAR ligand
Labeled fatty acids as ligands to PPAR were detected by
immunoprecipitating PPAR in pretreated cells and then detecting the
fatty acid attached to it by radio-TLC. A549 cells (1 x
106) were incubated with 0.25 µCi of
[14C]arachidonic acid (sp. act., 55 mCi/mmol;
Amersham International, Freiburg/Breisgau, Germany) for 24
h. After incorporation of the radioactive arachidonic acid (AA), cells
were washed and treated according to the experimental set-up (670 pM
IL-4 and 10 µM NDGA). Cell lysate was prepared and immunoprecipitated
with anti-PPAR Ab in 1 ml of RIPA buffer for 1 h at 4°C.
The immune complexes were allowed to bind to protein A/agarose (Santa
Cruz Biotechnology) for 1 h. The beads were spun down, washed
three times with RIPA buffer, and resuspended in 200 µl of PBS. This
solution was acidified with HCl to pH 3.5, and lipids were extracted
three times with ethyl acetate. After drying under a nitrogen stream
the sample was reconstituted in ethyl acetate and loaded onto a Silica
TLC plate (Merck, Darmstadt, Germany) and developed with
hexane/ether/acetic acid (50:50:0.01, v/v/v) as the solvent system. For
quantitation, the TLC plate was scanned on a radio-TLC scanner
(Berthold Instruments, Wildbad, Germany). AA; 5-, 12-, and 15-HETEs;
and various PGs were run on the side as standards.
Preparation of mitochondria
Cells were washed twice with PBS and trypsinized, and the cell pellet was collected. The pellet was resuspended in 5 vol of homogenization buffer (20 mM HEPES 7.5, 1.5 mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, and 250 mM sucrose) and homogenized for 5 min using a tight-fitting Dounce homogenizer (Kontes, Vineland, NJ). The effectiveness of the procedure was checked by a trypan blue exclusion test. Nuclei and cell debris were pelleted at 2,000 x g. Mitochondria were pelleted by centrifugation at 10,000 x g for 30 min at 4°C. The pellet was resuspended in RIPA buffer (see above). The supernatant was further centrifuged at 100,000 x g for 45 min at 4°C. The supernatant was used as the cytosolic extract. The protein concentration was determined in both mitochondrial and cytosolic fractions. Equal amounts of protein were used for additional experiments.
Statisics
Data are presented as the mean ± SD. Statistical comparisons between groups were made using Student’s t test for paired observations. Significance was achieved at the p < 0.05 level.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells treated with IL-4 and various inhibitors underwent
morphological changes and were examined for apoptosis. The TUNEL assay
showed that IL-4 (670 pM) induces apoptosis in A549 cells (Fig. 1A). Annexin V staining showed
similar results (Fig. 1B). Apoptotic cells stained green,
and necrotic cells stained red. This was further confirmed with a cell
death detection ELISA, in which cytoplasmic DNA-histone complexes were
detected (Fig. 2A). Moreover,
15(S)-HETE (30 µM) and 5 µM 15-deoxy-
12,14
PGJ2 (15-PGJ2), a PPAR
ligand, also caused apoptosis when preincubated for 72 h. Upon
preincubation of cells with 10 µM NDGA, a 15-LOX inhibitor,
IL-4-induced apoptosis was almost completely abolished (Fig. 2A). To determine whether this observation can be
extrapolated to other cell lines, human bronchial epithelial cells
(BEAS-2B) were treated in a similar manner and assayed for apoptosis by
the cell death detection ELISA. Indeed, the findings (Fig. 2B) confirmed that IL-4 and other effectors induced
apoptosis in BEAS-2B cells through a similar pathway as in A549 cells.
Consequently, additional experiments were confined to A549 cells.
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A549 cells were transiently transfected with PPAR-DN vector and
treated with 670 pM IL-4. IL-4-induced apoptosis was completely
abolished, indicating the prominent role of PPAR in the induction of
apoptosis (Figs. 2A and 3). In analogy, upon treatment of
PPAR-DN-transfected cells with 15-PGJ2 no
signs of apoptosis were observed (not shown)
15(S)-HETE is a ligand for PPAR in IL-4-stimulated A549 cells
In an earlier report we demonstrated that IL-4 treatment of A549
cells resulted in the up-regulation of 15-lipoxygenase, which, in turn,
augmented the production of 15(S)-HETE (22). It was
therefore of interest to determine whether 15(S)-HETE could serve as a
ligand for PPAR in this cell system. A549 cells were labeled with
[14C]AA and treated with 670 pM IL-4 for
72 h. Total protein extracts prepared from the cells were
subjected to immunoprecipitation with PPAR-Ab and protein A/agarose.
The lipids were extracted from the immune complex and analyzed by TLC.
In IL-4-treated cells a solitary radioactive lipid peak was observed.
It was identified as 15(S)-HETE by radio-TLC by cochromatography of
standard 15(S)-HETE (Fig. 4). Untreated
cells or cells treated with NDGA (10 µM) before IL-4 challenge failed
to show any radioactive ligand for PPAR. Moreover, IL-4 increased
PPAR-dependent promoter activity (Fig. 5), which could be inhibited by 10 µM
NDGA. Inasmuch as 15(S)-HETE was also observed to increase
PPAR-dependent promoter activity, an interaction between 15(S)-HETE
and PPAR receptors may be implicated. An analogous increase in
PPAR promoter activity was seen when cells were treated with 5 µM
15-PGJ2.
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up-regulates cleavage of caspase-8
Caspase-8 is one of the key upstream factors involved in the
up-regulation 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 compared with untreated cells or cells
treated with 10 µM NDGA before IL-4 induction (Fig. 6A). Similar up-regulation of
caspase-8 cleavage was observed with 5 µM PGJ2
and 30 µM 15-HETE. Caspase-8 cleavage was totally abolished in A549
cells transiently transfected with PPAR-DN vector (Fig. 6B).
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Death domain receptors are a family of cell receptors that
regulate the survival of the cell in response to various factors, such
as Fas ligand, TNF-
, and TRAIL. Upon activation, these receptors use
specific adapter proteins to activate the caspase-8 pathway. FADD is
such a vital adapter protein, which physically binds to caspase-8 and
is involved in the regulation of Fas- and TNF--mediated apoptosis
(30) To verify the involvement of death domain receptors
in IL-4- and PPAR-induced apoptosis in A549 cells, we transfected
A549 cells with a FADD-DN vector. This mutant lacks the death effector
domain, thus blocking the transmission of signal. As shown in Fig. 6B, IL-4-induced cleavage of caspase-8 was completely
blocked in cells transfected with FADD-DN vector. Moreover,
IL-4-induced cleavage of caspase-8, as measured by ELISA, was
completely abolished in cells transfected with FADD-DN (not shown).
These findings strongly suggest the involvement of death domain
receptors in IL-4- and PPAR-induced apoptosis in A549 cells.
Bid cleavage is not induced by caspase-8
The activated caspase-8 can stimulate apoptosis either via direct cleavage and activation of caspase-3 or by the mitochondrial route involving the cleavage of the C-terminal part of Bid, which then leads to release of cytochrome c. IL-4 and 15-PGJ2 induction failed to induce cleavage of Bid, as analyzed by Western blotting (Fig. 7). This suggests that caspase-8 may directly activate caspase-3 upon IL-4 treatment. However, determination of cytochrome c in the cytoplasmic fraction after challenging cells with IL-4 surprisingly revealed almost no release of cytochrome c. This implicates a cytochrome c-independent functional interplay between the pro- and anti-apoptotic members of the Bcl-2 family in IL-4-induced apoptosis in A549 cells.
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Bcl-xL is an anti-apoptotic 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 once released binds to apoptotic protease activating
factor-1 and leads to activation of caspase-3 via caspase-9. In
our hands, A549 cells treated with IL-4 showed a marked decrease in the
level of Bcl-xL, which could be completely
reversed by pretreating cells with NDGA as well as by transient
transfection with PPAR-DN vector (Fig. 8). The effects of IL-4 and PPAR
ligands on other members of the Bcl-2 family were also investigated. In
untreated cells, Bax, a pro-apoptotic member of the Bcl-2 family, was
predominantly present in the cytoplasm, but upon IL-4 treatment a large
amount of Bax was translocated to the mitochondria (Fig. 9). The decrease in
Bcl-xL (Fig. 8) with the concomitant activation
of Bax (Fig. 9) show the apoptosis blocking effect of
Bcl-xL through binding to Bax.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is predominantly
expressed in adipose tissue, where it plays a pivotal role in adipocyte
differentiation and lipid metabolism (25, 26). Upon
activation by ligand binding, PPAR heterodimerizes with retinoid X
receptor protein and binds to PPAR response elements present on the
promoters of various genes, thus regulating their function
(30). Most of these genes are involved in lipid metabolism
(26). The ligands for PPAR are mainly synthetic
anti-diabetic thiazolidinediones and 15-PGJ2
(28, 31), but also include a large number of
polyunsaturated fatty acids, among them arachidonic acid metabolites
such as 15(S)-HETE (24). PPAR transcription factors
have also been found in other tissues and blood cells, such as lung,
macrophages, and lymphocytes (24, 26). Upon stimulation of
macrophages with IL-4, up-regulation of PPAR-mRNA and protein
synthesis was observed (24).
PPAR receptors have been shown to regulate inflammation (32, 33) and
apoptosis in lung epithelial cells (34) and macrophages
(35). In the present study we have shown for the first time that
15(S)-HETE is bound as a ligand to PPAR transcription factors (Fig. 4) and is an effector of apoptosis (Figs. 2 and 3). Moreover, treatment
of cells with NDGA, a 15-lipoxygenase inhibitor, prevented PPAR
activation and apoptosis. This inhibition, however, could be almost
completely suppressed by the addition of 15(S)-HETE, thus ruling out
the possibility that the inhibition may be due to alteration of the
redox state of non-heme iron in the 15-LOX enzyme. Identical results
were also obtained with the PPAR ligand
15-PGJ2. Apoptosis was induced by a similar
mechanism in IL-4-treated human monocytes (not shown). To further
substantiate the crucial role of 15(S)-HETE in apoptosis via the
PPAR transcription factor, we used PPAR-DN, in which two amino
acids (L468A and E471A) have been mutated, thus impairing
transcriptional activation and cofactor recruitment (36).
Transfection of PPAR-DN in A549 cells strongly inhibited
IL-4-induced and 15-PGJ2-induced apoptosis (Figs. 2 and 3), supporting the prominent roles of 15(S)-HETE and 15
PGJ2 as effectors of apoptosis via PPAR
pathway. Experiments with normal bronchial epithelial cells (BEAS-2B)
confirmed the observations in A549 cells and thus underlined the
importance of these observations in human allergic inflammatory
reactions.
The exact downstream process of PPAR activation is still unclear.
Involvement of the death domain receptor in IL-4-induced apoptosis has
been observed in our experiments. Death domain receptors constitute a
family of cell surface receptors including CD95 (Fas/Apo-1), TNF-
receptor, DR3, DR4, and DR5 (37). Ligands initiate the
signaling cascade via receptor oligomerization and, through a set of
special adapter proteins, activation of the caspase cascade. Numerous
adapter proteins have been identified for these death receptors,
including FADD, TNFR1-associated death domain, receptor interacting
protein, and death-associated protein (38, 39, 40). The
FADD protein, which is common to all receptors and directly interacts
with caspase-8, consists of a death effector domain and a death domain
(41). The death domain interacts with caspase-8, which is
the next step in the cascade, while the death effector domain is
required in conjunction with other adapter proteins for interaction
with the receptors. The application of FADD-DN vector (42)
lacking the death effector domain abrogated the apoptotic signal
induced by IL-4 or PPAR (Fig. 6B). 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. 6A) This cleavage and activation are inhibited by NDGA and
PPAR-DN vector, demonstrating the vital importance of 15-LOX and
PPAR in the activation of apoptosis signal. Activated caspase-8 has
been proposed to stimulate apoptosis through two parallel pathways
(43, 44). In type I cells, capsase-8 directly cleaves and
activates caspase-3. Type II cells use 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. In our hands IL-4-treated A549 cells do not exhibit Bid
activation (Fig. 7), thus suggesting the involvement of the type I
pathway. It was previously shown that PPAR promotes TRAIL-induced
apoptosis (45). TRAIL uses various types of death
receptors, such as DR3, DR4, and DR5, to trigger apoptosis (46, 47). 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 cytochrome
c release (48). Inasmuch as cytochrome
c release is absent (Fig. 9), the dominating role for
apoptosis is apparently played by Bax, a proapoptotic Bcl-2-binding
protein. Thus, the scenario can be explained in the following way. In
type I cell death, binding of 15(S)-HETE to PPAR transcription
factor leads to generation of active caspase-8 through activation of
FADD protein within seconds (Fig. 6), which subsequently activates
downstream executioner caspase-3. In type II cell death, propagation
and amplification of the apoptotic signal require mitochondrial factors
and are therefore delayed. Due to the absence of cytochrome
c, we believe that a molecular link between caspase-3
activation and Bcl-xL is mediated by Bax. Bax may
bind to Bcl-xL, forming a heterodimer
Bcl-xL/Bax, in which Bcl-xL
dominates and prevents the induction of apoptosis (49). In
unstimulated A549 cells this pathway is blocked by
Bcl-xL, but, in IL-4-stimulated cells the binding
of 15(S)-HETE to PPAR transcription factor down-regulates
Bcl-xL (Fig. 8), and the resulting overexpression
of Bax commits the cell to apoptosis via caspase-3. Bax has also been
shown to be involved in a number of apoptotic pathways, especially the
DNA damage-induced apoptosis involving p53 (50), without
participation of death receptors. Anti-diabetic thiazolidinediones,
potent PPAR agonists, have been observed to induce apoptosis in
vascular smooth muscle cells through p53 and growth arrest and DANN
damage 45 pathways, although it is not clear whether PPAR
itself is the effector (51). In non-small cell lung cancer
cells, it has been shown that troglitazone induced the DNA
damage-inducible growth arrest and DANN damage 153 gene. Bax and p53
form an important pathway in DNA damage-induced apoptosis
(52). Caspase-3 has also been observed to activate the
mitochondrial apoptotic pathway by the cleavage of anti-apoptotic
Bcl-xL and Bcl-2 to pro-apoptotic components
(53, 54). Thus, caspase-3 activated by other pathways can
activate the mitochondrial route and provide a positive feedback loop
for caspase-3 production, leading to apoptosis.
In asthma, an up-regulation of IL-4 secretion in blood and higher
levels of 15-HETE in lung and bronchial tissue have been found. We
hypothesize therefore that IL-4-induced apoptosis is one of the major
causes of hypertrophy of the bronchial smooth muscle, denuded surface
epithelium, thickened basement membrane, and infiltration of
eosinophils, lymphocytes, and mononuclear phagocytes as well as the
apoptotic lesions observed in the lung tissue of asthma patients. It
has been reported that PPAR ligands induce apoptosis in lung cancer
cells, and this may be beneficial for the therapy of such cancers
(30). In contrast, in chronic inflammatory diseases such
as chronic obstructive pulmonary disease, the loss of alveolar
structures in the lung tissue due to apoptosis may worsen lung function
(29).
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Santosh Nigam, Eicosanoid and Lipid Research Division, Department of Gynecology, University Medical Center Benjamin Franklin, Free University Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany. E-mail address: nigam{at}zedat.fu-berlin.de
3 Abbreviations used in this paper: 5-HETE, 5-hydroxyeicosatetraenoic acid; 15-LOX, 15-lipoxygenase; AA, arachidonic acid; Ac-DEVD-pNA, acetyl-DEVD-para-nitroaniline; -DN, dominant negative; FADD, Fas-associating protein with death domain; NDGA, nordihydroguaiaretic acid; 15-PGJ2, 15-deoxy-12,14-PGJ2; PPAR, peroxisome proliferator-activated receptor ; Z-VAD-FMK, Z-Val-Ala-Asp(OMe)-CH2F.
Received for publication June 12, 2002. Accepted for publication October 29, 2002.
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 C. Liu, D. Xu, L. Liu, F. Schain, A. Brunnstrom, M. Bjorkholm, H.-E. Claesson, and J. Sjoberg 15-Lipoxygenase-1 induces expression and release of chemokines in cultured human lung epithelial cells Am J Physiol Lung Cell Mol Physiol, July 1, 2009; 297(1): L196 - L203. [Abstract] [Full Text] [PDF]
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U. Mabalirajan, A. K. Dinda, S. Kumar, R. Roshan, P. Gupta, S. K. Sharma, and B. Ghosh Mitochondrial Structural Changes and Dysfunction Are Associated with Experimental Allergic Asthma J. Immunol., September 1, 2008; 181(5): 3540 - 3548. [Abstract] [Full Text] [PDF]
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B. H. Maskrey, A. Bermudez-Fajardo, A. H. Morgan, E. Stewart-Jones, V. Dioszeghy, G. W. Taylor, P. R. S. Baker, B. Coles, M. J. Coffey, H. Kuhn, et al. Activated Platelets and Monocytes Generate Four Hydroxyphosphatidylethanolamines via Lipoxygenase J. Biol. Chem., July 13, 2007; 282(28): 20151 - 20163. [Abstract] [Full Text] [PDF]
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 Y. Torosyan, A. Dobi, S. Naga, K. Mezhevaya, M. Glasman, C. Norris, G. Jiang, G. Mueller, H. Pollard, and M. Srivastava Distinct Effects of Annexin A7 and p53 on Arachidonate Lipoxygenation in Prostate Cancer Cells Involve 5-Lipoxygenase Transcription Cancer Res., October 1, 2006; 66(19): 9609 - 9616. [Abstract] [Full Text] [PDF]
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 D. M. Simon, M. C. Arikan, S. Srisuma, S. Bhattacharya, L. W. Tsai, E. P. Ingenito, F. Gonzalez, S. D. Shapiro, and T. J. Mariani Epithelial cell PPAR{gamma} contributes to normal lung maturation FASEB J, July 1, 2006; 20(9): 1507 - 1509. [Abstract] [Full Text] [PDF]
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 M. Soller, A. Tautenhahn, B. Brune, K. Zacharowski, S. John, H. Link, and A. von Knethen Peroxisome proliferator-activated receptor {gamma} contributes to T lymphocyte apoptosis during sepsis J. Leukoc. Biol., January 1, 2006; 79(1): 235 - 243. [Abstract] [Full Text] [PDF]
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I. Komuro, T. Yasuda, A. Iwamoto, and K. S. Akagawa Catalase Plays a Critical Role in the CSF-independent Survival of Human Macrophages via Regulation of the Expression of BCL-2 Family J. Biol. Chem., December 16, 2005; 280(50): 41137 - 41145. [Abstract] [Full Text] [PDF]
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 F. R. Hirsch and S. M. Lippman Advances in the Biology of Lung Cancer Chemoprevention J. Clin. Oncol., May 10, 2005; 23(14): 3186 - 3197. [Abstract] [Full Text] [PDF]
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O. A. Skorokhod, M. Alessio, B. Mordmuller, P. Arese, and E. Schwarzer Hemozoin (Malarial Pigment) Inhibits Differentiation and Maturation of Human Monocyte-Derived Dendritic Cells: A Peroxisome Proliferator-Activated Receptor-{gamma}-Mediated Effect J. Immunol., September 15, 2004; 173(6): 4066 - 4074. [Abstract] [Full Text] [PDF]
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 D. Choudhary, I. Jansson, I. Stoilov, M. Sarfarazi, and J. B. Schenkman METABOLISM OF RETINOIDS AND ARACHIDONIC ACID BY HUMAN AND MOUSE CYTOCHROME P450 1B1 Drug Metab. Dispos., August 1, 2004; 32(8): 840 - 847. [Abstract] [Full Text] [PDF]
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 M. S. Shaik, A. Chatterjee, and M. Singh Effect of a Selective Cyclooxygenase-2 Inhibitor, Nimesulide, on the Growth of Lung Tumors and Their Expression of Cyclooxygenase-2 and Peroxisome Proliferator- Activated Receptor-{gamma} Clin. Cancer Res., February 15, 2004; 10(4): 1521 - 1529. [Abstract] [Full Text] [PDF]
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A. Tautenhahn, B. Brune, and A. von Knethen Activation-induced PPAR{gamma} expression sensitizes primary human T cells toward apoptosis J. Leukoc. Biol., May 1, 2003; 73(5): 665 - 672. [Abstract] [Full Text] [PDF]
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