Departments of 2 Neuroscience, 1 Biochemistry and Molecular Biology, and 3 Pediatrics, University of Florida, Gainesville, Florida 32610
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ABSTRACT |
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Cytokines are established regulators of the arachidonic
acid cascade in lung cells. The levels of various arachidonic
metabolites distinguish the normal and pathogenic states of the human
lung. Arachidonyl-selective cytosolic phospholipase A2
(cPLA2) is ubiquitously present in human lung and is most
likely the rate-limiting step in eicosanoid generation. We therefore
studied the regulation of this pivotal gene in human lung fibroblasts
and epithelial cells by proinflammatory cytokines. We demonstrate a
dose- and time-dependent induction of human cPLA2 mRNA by
interleukin-1, tumor necrosis factor-
, and interferon-
as well
as the abrogation of this induction by glucocorticoids. Nuclear runoff
studies demonstrate that de novo transcription of the
cPLA2 gene is required for cytokine
induction. We have characterized the human cPLA2 gene, which is encoded by 18 exons and spans in excess of 137 kb.
Deletion analysis of a 3.4-kb fragment of the human promoter identified
two regions responsible for basal expression of the cPLA2 gene. Conversely, a CA-dinucleotide repeat in
the proximal promoter appears to repress overall promoter activity. Understanding the molecular mechanisms associated with
cytokine-dependent expression of the cPLA2 gene
should provide further insight into regulating the level of
proinflammatory mediators in pulmonary diseases.
eicosanoids; gene regulation; gene structure
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INTRODUCTION |
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OF THE KNOWN BIOLOGICALLY active mediators generated by cells, both normally and during the inflammatory response, few have received more attention than those generated in the arachidonic acid cascade. Changes in the synthesis of eicosanoids have been shown to affect biological events as diverse as development, cellular differentiation, cell proliferation, and vascular homeostasis inflammation as well as apoptosis (1, 43). Further evidence of their importance is demonstrated by elevated levels of prostaglandin, leukotriene, hydroperoxyeicosatetraenoic acid, and platelet-activating factor in a number of diseases of the lung (22).
The rate-limiting step in the synthesis of proinflammatory eicosanoid metabolites has been ascribed to the enzymatic activity of the phospholipase A2 (PLA2) superfamily (12). This family of enzymes cleaves fatty acids located in the sn-2 position of a phospholipid, thereby releasing free fatty acid and lysophospholipid (3). The group IV cytoplasmic PLA2 (cPLA2) is an 85-kDa protein that has been shown to specifically hydrolyze arachidonic acid from the sn-2 position of phospholipids (10, 18). This arachidonic acid-selective event occurs after translocation of cPLA2 from the cytosol to the perinuclear membrane in response to agonist-dependent changes in intracellular calcium concentrations (17, 20).
Most recently, this enzyme has been implicated in numerous
physiological and pathophysiological processes as a central regulatory step in the synthesis of eicosanoid metabolites (3). cPLA2 enzyme activity as well as mRNA expression has been shown to be responsive to a number of stimuli including cytokines (23, 28, 46), growth factors (8, 32, 38), ATP (34), thrombin (19),
and glucocorticoid (14). Specifically, arachidonic acid release in
macrophages is induced by diverse agonists including tumor necrosis
factor (TNF)-, calcium ionophore, platelet-activating factor,
phorbol 12-myristate 13-acetate, okadaic acid, and the phagocytic
particle zymosan. Furthermore, the increase in arachidonic acid release
also correlates with the rapid activation of cPLA2 via
protein kinase-dependent enzyme phosphorylation (29, 34, 37, 42).
Although many studies have focused on the role of stimulus-dependent
phosphorylation and intracellular calcium fluxes on cPLA2 protein expression and activity, only a limited number of studies have
addressed the mechanisms by which cPLA2 gene
expression is controlled. Regulation at the transcriptional level may
relate to the delayed release of eicosanoids associated with the
resolution phase of the inflammatory response as well as to chronic
disease pathologies. Tay et al. (38) have shown that epidermal growth factor, platelet-derived growth factor, serum, and phorbol
12-myristate 13-acetate exert their effects on
cPLA2 expression through posttranslational mechanisms involving stabilization of the mRNA. Alternatively, a
transcriptional component has been attributed to the interferon (IFN)--mediated induction of cPLA2 in human
bronchial epithelial cells (46) and the glucocorticoid-dependent
repression of this gene in intestinal epithelial cells and fibroblasts
(14).
In relation to the human lung, cPLA2 mRNA was shown
to be far more ubiquitously expressed than secretory PLA2
(30). Protein expression is also regulated by cytokines in both human
lung fibroblast and bronchial epithelial cell lines (16, 28, 46). In
human WI-38 fibroblasts, interleukin (IL)-1 caused a threefold
induction in cPLA2 activity above that measured in control
cells. Likewise, in human bronchial epithelial (BEAS) cells,
cPLA2 activity, protein expression, and mRNA levels were
induced by the cytokine INF-. Fisher et al. (16) have
shown that TNF-
induces cPLA2 mRNA expression in
the BEAS cells as well. Therefore, due to the importance of
arachidonate metabolites in both normal and pathogenic lung function,
we set out to systematically define the cytokine-regulated expression
of cPLA2 in human lung cells by demonstrating that this
level of regulation occurs through de novo transcription. We have also
identified and characterized genomic clones encoding the human
cPLA2 gene. Although the human
cPLA2 promoter (30, 44) through exon 3 has
previously been identified (31), we have completed the characterization
of the entire human cPLA2 gene, which spans over
137 kb.
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METHODS |
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Materials. Recombinant human INF- and human
TNF-
were generously supplied by Genentech (San Francisco, CA).
IL-1
was a generous gift from Dr. Craig Reynolds (National Cancer
Institute, Bethesda, MD). Actinomycin D, cycloheximide,
lipopolysaccharide, and BSA were purchased from Sigma.
Random-primer DNA-labeling kit was purchased from GIBCO BRL (Life
Technologies). Zetabind and Noncharged Zetabind nylon membranes were
obtained from CUNO Laboratory Products. Hyperfilm-MP was purchased from
Amersham. The ABI PRISM dye terminator cycle sequencing core kit (with
AmpliTaq DNA polymerase) and the GeneAmp XL PCR
kit were purchased from Perkin-Elmer. Centri-Sep spin columns for use
with the cycle sequencing kit were purchased from Princeton
Separations. Nucleobond PC kit 500 plasmid DNA purification columns
were purchased from The Nest Group (Southborough, MA). Oligonucleotides
were synthesized by GIBCO BRL (Life Technologies).
Cell culture. A human fetal lung fibroblast cell line, IMR-90;
a human newborn lung fibroblast cell line, CCDLu-39; a human adenocarcinoma, epithelium-like lung cell line, A549; and a rat pulmonary epithelial cell line, L2, were purchased from American Type
Culture Collection. The pulmonary fibroblasts and epithelial cell lines
were cultured in MEM or Ham's F-12K medium, respectively, supplemented
with nonessential amino acids, L-glutamine (4 mM), penicillin G (100 U/ml), streptomycin (0.1 mg/ml), amphotericin B
(0.25 µg/ml), and 10% (vol/vol) fetal bovine serum. Confluent cell
lines were fed with their respective medium containing 1% fetal bovine
serum and cultured for an additional 12-18 h before treatments.
Unless otherwise indicated, the following reagents were used at these
final concentrations: 2 ng/ml of IL-1, 10 ng/ml of TNF-
, 25 U/ml
of human INF-
, 0.5 µg/ml of lipopolysaccharide, 5 µg/ml of
actinomycin D, and 20 µM cycloheximide.
RNA isolation, Northern analysis, and hybridization. Total
cellular RNA was isolated by cellular lysis in a guanidinium
isothiocyanate-sarcosyl solution followed by acid phenol-chloroform
extraction and ethanol precipitation as described previously (14).
Total RNA was size-fractionated on a 1% agarose-MOPS-formaldehyde gel
as described previously (14). After electrophoresis, the RNA was
electrophoretically transferred to a charged nylon membrane (Zetabind)
and ultraviolet cross-linked. Inserts containing coding sequences from
a human cPLA2 cDNA (generously provided by Dr.
James Clark, Genetics Institute, Cambridge, MA), a murine -actin
cDNA, and a rat cathepsin B cDNA were used to generate radiolabeled
probes for Northern analyses. Probes were synthesized with
[
-32P]dATP by random-primer extension.
Membranes were hybridized as previously described (14) followed by
autoradiography. All depicted Northern analysis data portray a
representative study from a minimum of three independent experiments.
Nuclear runoff assay. Analysis of nascent radiolabeled mRNA
transcripts from control and cytokine-treated CCDLu-39 and IMR-90 cells
was performed according to a modified procedure described previously
(27). Nascent RNA transcript elongation from an equal number of control
and treated nuclei (3-4 × 107) was carried out
in the presence of 250 µCi of [-32P]UTP at
30°C for 45 min. The reaction was terminated, and transcription rates were analyzed in hybridization solution containing radiolabeled RNA (1-2 × 107 counts/min) from control and
IL-1
-treated cells with strips of nylon membrane blotted with
denatured cPLA2 coding region insert and various
control DNA fragments.
Immunoblot analysis. CCDLu-39 cells were grown as described in
Cell culture and treated with 2 ng/ml of IL-1
for 12, 18, 24, and 36 h, and the total cell lysate was isolated by
homogenization in 10 mM Tris-Cl, 1 mM EDTA, 250 mM sucrose, 35 µg/ml
of phenylmethylsulfonyl fluoride, and 1% Triton X-100. Total protein
was determined by a modified Bradford assay (Pierce). Fifty micrograms
of protein were loaded on a 12.5% SDS-polyacrylamide gel, and proteins
were fractionated for 16 h at 50 V. The gels were electrotransferred to
nitrocellulose and incubated with a 1:100 dilution of a primary monoclonal antibody IgG against cPLA2 (Santa Cruz
Biotechnology) in Tris-buffered saline, 0.1% Tween, and 5% nonfat dry
milk for 1 h. The membranes were washed in the same buffer and then
incubated with a goat anti-mouse antibody (IgA, IgG, or IgM) coupled to horseradish peroxidase (Kirkegaard and Perry Laboratories) followed by
detection with an Amersham enhanced chemiluminescence protocol. Membranes were exposed to Amersham Hyperfilm, and the levels of expression were quantitated with National Institutes of Health Image software.
Characterization of human P1 and PAC cPLA2 genomic clones. Human cPLA2 P1 (2455 and 2456), PAC (9380 and 9381), and BAC (215:G22) clones were obtained from library screenings conducted in conjunction with Genome Systems (St. Louis, MO). The initial P1 library screening was completed in collaboration with Dr. James Clark. P1, PAC, and BAC clones were amplified, and DNA was purified according to the Nucleobond-AX 500 protocol with a modified alkaline lysis method of Birnboim and Doly (2), followed by purification on a Nucleobond anion-exchange column. To determine the boundaries of each clone, restricted P1, PAC, or BAC DNA or XL-PCR products were size-fractionated on 1% agarose gels, and oligonucleotides corresponding to various regions of the coding sequence were used as probes for Southern analyses. Fluorescence-based cycle sequencing reactions were carried out on high-quality DNA preparations of P1 and PAC human cPLA2 genomic clones with an ABI PRISM dye terminator cycle sequencing core kit with AmpliTaq DNA polymerase. Oligonucleotides (24-29 bases) corresponding to the human cPLA2 cDNA were synthesized to serve as primers for the sequencing reactions.
Long-range PCR. A long-range PCR procedure (7) was used to amplify human cPLA2 introns from P1, PAC, or BAC clones and human genomic DNA with a GeneAmp XL PCR kit. Briefly, 12 PCR cycles were followed by an incremental lengthening (15 s) of the extension time for an additional 16-22 cycles and a final 15-min, 72°C extension step. Concentrations of template, magnesium, and polymerase as well as PCR conditions (primarily annealing temperature) were varied to increase yield of the specific products. Long-range PCR products were size-fractionated on agarose gels to estimate intron sizes.
Transient transfection assays. A 3.4-kb human promoter fragment
was obtained by long-range PCR with human promoter sequence (GenBank
accession no. D38178) reported by Morri et al. (31). This
promoter was cloned 5' to the human growth hormone (hGH) reporter gene of the promoterless pGH vector (35). Promoter deletions of a 3.4-kb human promoter fragment were transiently transfected in CCDLu-39 human lung fibroblast cells with a modification of the DEAE dextran-mediated transfection method (26) or with Superfect
(Qiagen). The transfected cells were incubated for 24 h at 37°C and
5% CO2 in air. Each 15-cm plate of cells was trypsinized, passed to eight 60-mm plates, and grown overnight. At 70-90%
confluency, various pro- and anti-inflammatory agents were added to the
transfected cells. Forty-eight hours later, medium was sampled from
each plate and stored at
75°C for hGH assay. The hGH
concentration in a 100-µl sample of medium was measured with the hGH
assay kit (Allegro; Nichols Institute).
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RESULTS |
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Cytokine effects on cPLA2 expression in human lung
fibroblasts and epithelial cells. The regulated expression of the
cPLA2 gene in human pulmonary fibroblast and
epithelial cell lines was examined in light of the involvement of these
cells in both eicosanoid production and the inflammatory process.
Representative Northern analyses in Fig. 1,
A and B, demonstrate regulated
cPLA2 expression in CCDLu-39 and IMR-90,
respectively, two human lung fibroblast cell lines, after 12-h
treatments with various proinflammatory mediators. IL-1, TNF-
,
and INF-
elicited ~12-, 8-, and 6-fold induction, respectively, in
cPLA2 mRNA levels relative to that in unstimulated
cells. On the other hand, transforming growth factor-
, an
established mediator of collagen synthesis in lung fibroblasts, did not
alter cPLA2 mRNA levels in either cell line, which
concurs with a previous report (24).
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Epithelial cells have been identified as active participants in both arachidonate metabolism and the inflammatory processes within the lung (22); therefore, we also evaluated cytokine-dependent expression of cPLA2 in human lung epithelial cells. Figure 1C illustrates a more modest response in cPLA2 mRNA expression to cytokines in A549 cells, a human adenocarcinoma alveolar type II-like cell line. Therefore, on the basis of this marked difference in the cytokine-dependent induction of cPLA2 mRNA levels in the human lung fibroblast cells, as well as the importance of arachidonate metabolites in lung fibroblast activity, it was of interest to further delineate the cytokine regulation of cPLA2 in these cells.
Dosage effects of cytokines on cPLA2 gene expression.
To evaluate the physiological relevance of the cytokine response,
the effects of increasing IL-1 (Fig.
2A) and TNF-
(Fig.
2B) concentrations on cPLA2 mRNA levels in
CCDLu-39 cells were evaluated at 12 h. A half-maximal induction in
cPLA2 mRNA levels occurred between 0.1 and 1.0 ng/ml for TNF-
and 20 and 200 pg/ml for IL-1
. As a consequence of
these results, all subsequent treatments in this study employed 10 ng/ml of TNF-
and 2 ng/ml of IL-1
.
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Kinetics of IL-1 and TNF-
induction of
cPLA2 mRNA and protein expression. Figure
3A illustrates a time-dependent
induction of cPLA2 mRNA levels in CCDLu-39 cells in
response to IL-1
treatment. Induced mRNA expression was observed as
early as 2 h, with a maximal effect on cPLA2
mRNA levels attained between 8 and 12 h. Densitometric analysis
estimated a 12-fold increase in steady-state cPLA2
mRNA expression over the control value at 12 h. Figure 3B
exemplifies similar kinetics for TNF-
-mediated
cPLA2 mRNA induction, with an estimated eightfold
increase over basal message levels. Similar kinetics of both IL-1
-
and TNF-
-mediated increases of cPLA2 steady-state
message levels were observed in IMR-90 human fetal lung fibroblasts
(data not shown). To demonstrate that increases in mRNA levels result
in concomitant increases in cPLA2 protein levels, CCDLu-39
cells were treated with IL-1
and cPLA2 protein detected
by immunoblot analysis. Figure 3C shows a representative immunoblot experiment, and Fig. 3D illustrates a densitometric analysis of independent experiments. The data demonstrate that treatment with IL-1
results in an approximately fourfold
increase in cPLA2 protein levels.
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Effect of transcriptional and translational inhibitors on
cPLA2 cytokine response. In Fig.
4, left, cotreatment of CCDLu-39 cells with IL-1 and actinomycin D blocked cytokine-induced
cPLA2 mRNA expression. TNF-
in conjunction with
actinomycin D causes cellular cytotoxicity or possibly apoptosis in
both CCDLu-39 (Fig. 4) and IMR-90 (data not shown) cells as illustrated
by decreased expression of actin mRNA under these experimental
conditions. We would therefore cautiously interpret the inhibition of
TNF-
induction of cPLA2 as requiring a
transcriptional component.
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To determine whether the cytokine-mediated induction of
cPLA2 mRNA requires a translational event(s),
CCDLu-39 cells were treated with the protein synthesis inhibitor
cycloheximide (20 µM) in the presence and absence of either IL-1
or TNF-
(Fig. 4, right). At 6 h, cycloheximide treatment
caused an induction of basal cPLA2 mRNA levels.
This effect on basal mRNA levels suggests that a protein(s) may be
involved in the degradation of the cPLA2 message. However, cycloheximide did attenuate cytokine induction of
cPLA2 mRNA, implying that a translation-dependent
mechanism may be associated with both IL-1
- and TNF-
-dependent
increases in cPLA2 expression. These inhibitor
studies imply that in addition to ongoing protein synthesis a
transcriptional component is essential to the cytokine-mediated
induction of cPLA2 mRNA expression.
Cytokine effect on nascent cPLA2 RNA synthesis.
Recently, INF- has been shown to directly regulate the
transcription rate of cPLA2 in a human bronchial
epithelial cell line (46). Therefore, to assess whether IL-1
and
TNF-
induction of cPLA2 steady-state mRNA levels
reflect a direct increase in cPLA2 gene
transcription, a nuclear runoff assay was employed (27). Figure
5 illustrates that radioactive UTP
incorporation into nascent cPLA2 mRNA was elevated
in IL-1
-stimulated IMR-90 cells over that in control cells relative
to
-actin nuclear RNA transcript levels. Therefore, the IL-1
induction of cPLA2 mRNA seen at the steady-state
level is due in part to de novo cPLA2 mRNA
synthesis. Nuclear runoff assays were also conducted on IL-1
- and
TNF-
-stimulated CCDLu-39 cells, with similar results (data not
shown).
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Glucocorticoid abrogates cytokine induction of cPLA2
gene expression. Previous studies from our laboratory (14) and
others (21, 25, 28) have shown that cytokine induction of
cPLA2 mRNA expression can be blocked by
glucocorticoid treatment in several distinct cell types. Figure
6 illustrates that cotreatment of
cytokine-stimulated human lung fibroblast cells (CCDLu-39) with
glucocorticoid results in mitigation of the observed cytokine-mediated inductions of cPLA2 mRNA by 12 h. In light of the
pivotal role cPLA2 plays in the generation of
proinflammatory eicosanoid metabolites, the anti-inflammatory benefits
associated with steroid therapy in the treatment of human pulmonary
disorders may in part involve decreased expression of
cPLA2.
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Characterization of human P1, PAC, and BAC genomic clones encoding
the human cPLA2 gene. Given the physiological
importance of cPLA2 to the inflammatory process in the lung
and the demonstration that cytokine and glucocorticoid regulation is
dependent on de novo gene regulation, we initiated studies to
characterize the human cPLA2 genomic locus. Human
P1, PAC, and BAC genomic libraries (36) were screened (Genome Systems)
with oligonucleotide primer sets generated from cDNA, published
5' noncoding, and exon 1 sequence, which corresponded to
single-copy genomic sequence. Five overlapping clones were identified
(one BAC, two P1, and two PAC) that hybridize to a known
cPLA2 sequence. To delineate the regions of the
human cPLA2 gene that each of these genomic clones
encompassed, Southern analysis was performed on restriction enzyme
digests of P1, PAC, and BAC DNA clones with radiolabeled
oligonucleotides corresponding to discrete sequences along the
cPLA2 cDNA. This initial screening technique
determined that the five genomic clones overlapped by cumulatively
encoding the full length of cPLA2 mRNA over 20 kb
of a 5'-flanking sequence and a large region of a
3'-noncoding sequence (summarized in Fig. 7B).
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The strategy used to assign exon/intron boundaries to the human cPLA2 gene is outlined in Fig. 7A. For example, primer A in Fig. 7A was used to define the 3' boundary of exon 4 by cycle sequencing of the respective genomic clones, with primer B, corresponding to the bottom strand sequence, providing the 5' boundary of exon 4. These boundaries were confirmed with primers corresponding to adjacent exon sequence (i.e., exon 3, primer A' and exon 5, primer B'). With this strategy, the 2.9-kb human cPLA2 cDNA was found to be derived from 18 exons ranging in size from 37 (exon 6) to 612 bp (exon 18), the latter encoding the extensive 3'-untranslated region of this message (Fig. 7B). All intron/exon junctions were consistent with the consensus sequences corresponding to human intron donor/acceptor splice sites (GT/AG rule).
With knowledge of the intron/exon boundaries, opposite strand primers
corresponding to adjacent exons were used to amplify the intronic
fragment with long-range PCR (Fig. 7A) (7). These long-range
PCRs were size-fractionated on an agarose gel to determine the size of
the corresponding introns, which are summarized along with exon sizes
in Table 1. Combining the data generated
from cycle sequencing and long-range PCR experiments as well as from intron sizes previously reported (31), the cPLA2
gene occupies a 137-kb stretch of DNA on human chromosome 1q. A
schematic representation of the human cPLA2 genomic
structure designating the approximate positions of the BAC, P1, and PAC
genomic clones as well as the relative positions of the 18 exons and
introns is shown in Fig. 7B.
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cPLA2 promoter deletion analysis. Given the
availability of the cPLA2 promoter from the genomic
clones and the demonstration that cytokine- and
glucocorticoid-dependent (14) induction requires de novo transcription,
we attempted to delineate cis-acting sequences involved in
basal human cPLA2 gene expression using transient transfection of promoter/reporter gene constructs. A 3.4-kb fragment of
the human cPLA2 promoter was inserted into a
hGH reporter vector as well as into a series of promoter
deletions. These constructs were transiently transfected into a number
of cell lines, and secreted hGH levels were measured to assay
promoter activity. Figure 8
represents the basal hGH expression in transfected CCDLu-39 fibroblasts normalized relative to the longest promoter fragment, with
similar results found in IMR-90 and A549 cells. Deletion of 1,175 bp
from positions 3446 to
2271 of the 5'-flanking
region caused a significant reduction in basal promoter activity.
Further deletion from
543 to
215 binding sites also
resulted in a significant reduction in reporter activity. Previously,
Miyashita et al. (30) and Wu et al. (44) evaluated deletions of a
minimal ~500 bp cPLA2 promoter construct and
observed only the latter loss of promoter activity. Additionally, Wu et
al. identified a CA repeat centered around position
220 as a
potential negative regulatory element that when deleted from our 3.4-kb
promoter construct caused a 40-50% increase in promoter activity
(Fig. 8). Similar to previous results (30, 44) with a minimal
cPLA2 promoter, our 3.4-kb promoter was not
adequate to confer a stimulus-dependent increase in reporter activity,
implying that regulatory elements specific to IL-1
and TNF-
induction are located outside this sizable promoter region.
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DISCUSSION |
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Both immune cells and cells localized to the alveolar wall are well-established participants in the inflammatory response that characterizes the initial phase of many pulmonary diseases. The lung epithelial layer is a common site of injury, and these cells are active participants in triggering immune cell activation and infiltration. Resident cells and immune cells produce numerous soluble factors including cytokines, chemokines, and mitogenic factors as well as arachidonic acid metabolites that are necessary for the control and potentiation of the inflammatory response. Conversely, fibroblasts have traditionally been associated with the fibrotic and tissue remodeling phase of the disease through proliferation and deposition of extracellular matrix. Elias and Kotloff (15), however, have demonstrated that lung fibroblasts can actively participate in establishing the chronic inflammatory state associated with lung fibrosis as effector cells. Fibroblasts can contribute to the synthesis of numerous proinflammatory molecules such as interleukins as well as arachidonate metabolites.
In the lung, eicosanoids have been associated with inflammation, fibrosis (11), pulmonary hypertension (9), and asthma (13). Although high levels of PGE2 in the healthy lung are associated with both suppression of collagen production and decreased rate of mitogen-stimulated fibroblast proliferation, the levels of PGE2 in the epithelial lining of the fibrotic lung are significantly decreased (11). Most recently, two classes of leukotrienes have been associated with the pathobiology of asthma that have led to the effective use of 5-lipoxygenase inhibitors and receptor antagonists in the treatment of asthma, with improved pulmonary function and reduced bronchoconstriction. In addition to their proinflammatory role, including vasoconstriction, vasodilation, and chemoattractive functions in the lung, arachidonic acid metabolites also participate in tissue remodeling associated with pulmonary dysfunctions such as fibrosis.
The 85-kDa cPLA2 is the first arachidonic acid-selective PLA2 to be identified and is now recognized as a key enzyme in mediating agonist-induced arachidonic acid release. Its relevance to lung biology has been strikingly demonstrated in recent studies (4, 41) with the ablation of the cPLA2 gene. These mice exhibit a significant reduction in ovalbumin-induced anaphylactic responses as well as a dramatically diminished bronchial reactivity to methacholine challenge (41). Therefore, given the current effectiveness of antileukotriene therapy in asthma and the results of gene ablation, cPLA2 may play a central role in lung pathogenesis.
cPLA2 activity is regulated by both transcriptional and
posttranscriptional mechanisms. The acute or rapid accumulation of arachidonate derived from cPLA2 activity results from
activation of preexisting enzyme through phosphorylation (29, 34, 37, 42). In this report, we have assessed the molecular regulation of
cPLA2 by TNF- and IL-1
in human lung fibroblast and
epithelial cells. We have demonstrated a dose- and time-dependent
induction of steady-state cPLA2 mRNA levels in
response to TNF-
and IL-1
exposure. In both lung fibroblasts and
epithelial cells, cytokine-dependent induction of
cPLA2 mRNA levels occurred within 2 h, with a
maximal expression at 12 h estimated to be 12-fold for IL-1
, 8-fold
for TNF-
, and 6-fold for INF-
. Most importantly, we have
demonstrated that this induction requires de novo transcription and
possibly new protein synthesis. This induction is also accompanied by a concomitant fourfold increase in cPLA2 protein levels in
response to IL-1
exposure. Increased cPLA2 mRNA
transcription and protein synthesis is consistent with studies in
bronchial epithelial cells (45), 3T3 fibroblasts (46), human
fibroblasts (28), and rheumatoid synovial fibroblasts (23), which have
demonstrated an increase in cPLA2 enzyme activity after
cytokine stimulation.
Current therapy for many pulmonary diseases includes the use of corticosteroids. The therapeutic benefits associated with steroid treatment are in part a consequence of the inhibitory effects on eicosanoid biosynthesis, where the release of free arachidonate through the action of PLA2 is the rate-limiting step. Dolan-O'Keefe and Nick (14) have previously shown that basal cPLA2 activity and mRNA levels are repressed by glucocorticoids and that, as with cytokines, de novo transcription is required for steroid repression. We have extended these findings by demonstrating that cytokine induction of cPLA2 mRNA levels in pulmonary cells can be effectively repressed by cotreatment with glucocorticoids, thus in part offering a rationale for the anti-inflammatory effects of steroids.
To further study cPLA2 gene regulation, we have characterized the entire genomic structure of the human cPLA2 gene. The gene spans a >137-kb region of the human genome and is composed of 18 exons. Given the large size of this gene, traditional methodologies of mapping genomic clones, which entail subcloning, restriction mapping, and sequencing, were labor intensive and impractical. Therefore, we developed an alternative approach to characterize this large gene whereby positive BAC, P1, and PAC clones were characterized with both cycle sequencing and long-range PCR techniques. This approach allowed the identification of intron/exon boundaries as well as the size and location of each of the introns relative to our genomic clones (Fig. 7B, Table 1).
With the availability of these genomic clones and evidence that
cytokine-dependent regulation required de novo transcription, we have
identified regions of the cPLA2 promoter that are
involved in basal gene expression. Previously, constitutive cPLA2 promoter activity has been detected in both a
human laryngeal epidermoid cell line, HEp-2 (30), and a human bronchial epithelial cell, BEAS-2B (44), when transfected with a construct encoding ~500 bp of the 5'-flanking region of the human
cPLA2 gene. To extend these studies, we evaluated a
3.4-kb cPLA2 promoter fragment for
cis-acting regulatory sequences. These studies identified two
regions, between binding sites 3446 and
2271 and between
binding sites
543 and
215, that reduce promoter activity
when deleted. In addition, Wu et al. (44) had previously noted the
presence of a 48-bp CA-dinucleotide repeat in the
cPLA2 promoter ~150 bp upstream from the
transcriptional start site that is conserved in both the human and rat
genes (31). This CA repeat is found in the 5'-flanking region of
many genes (40) and has been postulated to confer regulatory effects on
gene transcription by adopting a Z-DNA conformation. A CA
repeat located in the promoter of the rat prolactin gene has been shown
to be associated with transcriptional repression (33), with similar
results reported for the proto-Ha-ras gene (6). Although
transfection of a cPLA2 promoter containing the CA
repeat in either the human HEp-2 cells (30) or the rat mesangial cells
(39) had no effect on reporter gene expression, a similar transfection
study in human BEAS cells showed that this dinucleotide repeat might be
associated with suppression of cPLA2 transcription
(44). Deletion of the CA repeat from our 3.4-kb promoter resulted in a
40-50% increase in promoter activity, further substantiating the
potential role of this sequence in the inhibition of gene transcription.
In conclusion, given the central importance of arachidonic acid
metabolites in pulmonary disease pathologies, IL-1, TNF-
, and
INF-
induction of cPLA2 gene expression in human lung fibroblasts (15) and epithelial cells (5) may be an important
contributing factor in lung inflammation. Moreover, because this
cytokine-dependent induction requires de novo gene transcription and
can be strongly attenuated by cotreatment with dexamethasone,
strategies to regulate cPLA2 gene expression should
offer a logical therapeutic regimen to control proinflammatory levels
of arachidonate metabolites. Further studies to elucidate the precise
regulatory elements controlling cytokine- and glucocorticoid-dependent regulation are currently in progress and should aid in identifying mechanisms to control cPLA2 expression as therapy
for a variety of pulmonary diseases.
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ACKNOWLEDGEMENTS |
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We thank Karen Ackey (Center for Mammalian Genetics Core, Gainesville, FL) for technical assistance with sequence analysis and Sean O'Keefe for help with statistical analysis.
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FOOTNOTES |
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This work was supported in part by an American Lung Association grant (to H. S. Nick).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. S. Nick, Dept. of Neuroscience, PO Box 100244 JHMHC, Univ. of Florida, Gainesville, FL 32610-0245 (E-mail: hnick{at}ufl.edu).
Received 5 April 1999; accepted in final form 26 October 1999.
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