From the Program in Human Molecular Biology and
Genetics, the Huntsman Cancer Institute, and the § Nora
Eccles Harrison Cardiovascular Research and Training Institute,
University of Utah, Salt Lake City, Utah 84112
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ABSTRACT |
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Platelet-activating factor (PAF) is a
potent phospholipid with diverse physiological and pathological
actions, and it is inactivated by PAF acetylhydrolase. In this study,
we analyzed the tissue distribution of the plasma PAF acetylhydrolase
mRNA in humans. We isolated a 3.5-kilobase fragment containing the
5' genomic sequence of the plasma PAF acetylhydrolase gene and further
characterized the promoter activity. We determined the transcriptional
initiation site by primer extension. We then prepared constructs
containing various lengths of 5' genomic fragments fused to a
luciferase reporter gene and transfected these constructs into COS-7
cells. We found that there is more than one region in the 1.3-kilobase 5' genomic sequence conferring promoter activity and that a very short
5'-flanking region (72 base pairs) is sufficient for more than 65% of
the basal activity. In parallel, we examined the regulation of
expression of the PAF acetylhydrolase gene. We found that
interferon- (IFN
) and lipopolysaccharide (LPS) significantly
inhibited synthesis of PAF acetylhydrolase, whereas other
cytokines, including IFN
, interleukin (IL) 1
, IL4, IL6, tumor
necrosis factor-
, granulocyte/macrophage colony-stimulating factor,
and macrophage colony-stimulating factor, had a smaller or no effect in
human monocyte-derived macrophages. Furthermore, transfection of the
promoter/reporter construct into macrophage RAW264.7 cells revealed
that IFN
and LPS decreased the promoter activity by 35% and 50%,
respectively, whereas PAF stimulated it by 52% via its receptor. The
promoter activity was much lower in monocytic U937 cells compared with
the basal level in COS-7 cells, while the activities in P388D1 and
RAW264.7 macrophagic cells were considerably higher than the basal
level in COS-7 cells. There are multiple regions in the PAF
acetylhydrolase promoter that contain responsive elements for signal
transducer and activators of transcription-related proteins, and also
for myeloid-specific transcription factors. Our data indicate that the
opposite of mRNA expression in monocytes versus
macrophages is due to inhibition of the promoter activity in the former
and activation in the latter cells.
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INTRODUCTION |
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Platelet-activating factor (PAF,1 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent phospholipid with diverse physiological and pathological effects in a variety of cells and tissues (for review, see Refs. 1-3). The biological effects of PAF include activation of platelets, polymorphonuclear leukocytes, monocytes, and macrophages. PAF also increases vascular permeability, decreases cardiac output, induces hypotension, and stimulates uterine contraction (2). The actions of PAF may contribute to the physiological processes associated with pregnancy, fetal development, parturition, and kidney function. PAF has been implicated in pathological processes, such as inflammation and allergy (4). Moreover, there are indications that PAF might be involved in human immunodeficiency virus pathogenesis and in carcinogenesis (5, 6).
The levels of PAF in plasma and tissues are determined by the balance of synthesis and degradation (7). A key mechanism for the removal of PAF is hydrolysis catalyzed by PAF acetylhydrolase, which converts PAF to the biologically inactive lyso-PAF (8). This enzymatic activity has been detected in plasma and in the cytosolic fraction of some cells and tissues (9, 10). Molecular studies of the PAF acetylhydrolase from various sources indicate that the plasma and cytosolic forms of this enzyme are encoded by different genes (11-13). Several lines of evidence indicate that monocyte-derived macrophages are a major source of plasma PAF acetylhydrolase. Messenger RNA for the plasma PAF acetylhydrolase is induced and a large amount of this activity is secreted when human monocytes differentiate to macrophages (11, 14). Furthermore, the properties of the macrophage-secreted PAF acetylhydrolase are identical to those of the plasma form, and the secreted enzyme is recognized by an antibody raised against the purified plasma PAF acetylhydrolase (15). Thus, monocyte-derived macrophages clearly secrete the plasma form of PAF acetylhydrolase. Tjoelker et al. (11, 16) cloned the plasma PAF acetylhydrolase cDNA by screening a human macrophage cDNA library. The encoded protein is composed of 441 amino acid residues with a catalytic triad GXSXG, which is conserved in neutral lipases and serine esterases.
Plasma PAF acetylhydrolase dramatically reduces the inflammatory action of PAF both in vitro and in vivo, suggesting that this enzyme plays an important role in the regulation of PAF levels in blood and tissues (11). Moreover, PAF acetylhydrolase also catalyzes the hydrolysis of oxidatively fragmented phospholipids, which have been detected in oxidized low density lipoprotein and have been shown to be mitogenic and inflammatory under certain circumstances (17). Therefore, PAF acetylhydrolase may protect against oxidative damage.
The secretion of PAF acetylhydrolase activity has been shown to be
regulated by many cytokines and hormones in cultured macrophages and
hepatocytes. Previously, we showed that HepG2 cells and primary cultures of hepatocytes secrete PAF acetylhydrolase activity (although at relatively low levels compared with macrophages) (18). We found no
substantial effect of dexamethasone on the secretion of this activity
by hepatocytes, while Narahara et al. (19) showed that
dexamethasone increases secretion from HL-60 cells that have
differentiated to macrophages. In addition, lipopolysaccharide (LPS),
interleukin (IL) 1, IL1
, and tumor necrosis factor-
(TNF
)
reduced the secretion from decidual macrophages (20). It was observed
that estrogen administration to animals decreased PAF acetylhydrolase
secretion (21). In in vitro studies, however, the effect
depended on the cell type; estrogen reduced the secretion from HepG2
cells but had no effect on differentiated HL-60 cells (18, 19). Another
regulator of PAF acetylhydrolase expression is its substrate. Satoh
et al. (22) showed that PAF, but not the product of the
reaction, lysoPAF, stimulated the secretion of PAF acetylhydrolase from
HepG2 cells. In summary, previous studies have shown that macrophages
secrete high levels of PAF acetylhydrolase activity, which can be
influenced by inflammatory mediators. However, the regulatory mechanism
was not examined in any of the published studies.
One of the most dramatic examples of regulation of PAF acetylhydrolase synthesis is during the differentiation from monocytes to macrophages. Low levels of PAF acetylhydrolase mRNA were detected in isolated human monocytes by Northern analysis. In contrast, large amounts were detected once these cells differentiated to macrophages (11). This agreed with a previous report, in which the enzymatic activity was shown to increase during the maturation process (15). PAF acetylhydrolase mRNA also was expressed differentially among tissues; it was highly expressed in tonsil and placenta but not in a number of others (11). Taken together, the above results strongly suggest that plasma PAF acetylhydrolase is regulated in a tissue- and differentiation-dependent manner.
We report here the cloning of a 3.5-kb 5' genomic fragment of the
plasma PAF acetylhydrolase gene. We demonstrate that this fragment
exhibits various levels of promoter activity in monocytic and
macrophagic cell lines, and characterized the basic features of this
promoter. Furthermore, we found that LPS and interferon (IFN
)
inhibit the promoter activity while PAF stimulates it.
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EXPERIMENTAL PROCEDURES |
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Materials and Cell
Lines--
Hexadecyl-2-[3H-acetyl]-sn-glycero-3-phosphocholine
was purchased from NEN Life Science Products.
1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) was
obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). All chemicals
and reagents were from Sigma unless otherwise specified. All of the
human and murine cytokines were from R&D Systems. PAF antagonists
BN52021 and CV3988 were obtained from Biomol (Plymouth Meeting, PA).
[-32P]ATP (10 mCi/ml) was obtained from Amersham Life
Science. Cell culture reagents and restriction enzymes were purchased
from Life Technologies, Inc. except where specified. The monkey kidney
cell line COS-7, human monocytic cell line U937, and mouse macrophage cell lines RAW264.7 and P388D1 were from American Type Culture Collection (Rockville, MD). The human embryonic kidney cell line 293 that was stably transfected with the PAF receptor, and the rabbit
anti-plasma PAF acetylhydrolase antibody were provided by ICOS Corp.
(Bothell, WA). The mouse monoclonal antibody against actin was from ICN
Biomedicals, Inc. (Costa Mesa, CA).
Isolation of a PAF Acetylhydrolase Genomic Clone--
A human
genomic library (Stratagene, La Jolla, CA) was screened with a
32P-labeled SmaI-KpnI cDNA
fragment containing 277 bp of the 5' end of the cDNA sequence. The
probe was labeled with [
-32P]dCTP by random priming,
and hybridization was carried out at 42 °C for 16 h. Positive
plaques were detected by autoradiography. The
DNA insert in one of
the positive plaques was subcloned into pBluescript (SK), and Southern
analysis was performed to identify regions containing exon 1. A 3.5-kb
NotI-BamHI genomic fragment hybridized with the
exon 1 probe, and then was subcloned into pBluescript and termed
pDS.
mRNA Dot Hybridization-- A human RNA master blot was purchased from CLONTECH. The amount of poly(A)+ RNA from different tissues on this blot was normalized for eight housekeeping genes by the manufacturer. A human plasma PAF acetylhydrolase cDNA clone (11) was linearized by SmaI digestion, and the 2.3-kb fragment was purified and used as template for RNA probe preparation. The antisense RNA was synthesized and labeled with the DIG RNA-labeling kit (Boehringer Mannheim) by an in vitro transcription reaction. The generated 1.2-kb antisense RNA probe then was hybridized to the blot following the manufacturer's instructions. The levels of PAF acetylhydrolase mRNA were quantified by scanning using Photoshop software and analyzed using NIH Image.
Primer Extension-- The 5' end of an antisense oligonucleotide RAH+40, was labeled using T4 polynucleotide kinase (Promega, Madison, WI) and purified through a NucTrap probe purification column (Stratagene). The primer extension reaction was performed using an AMV (avian myeloblastosis virus) reverse transcriptase primer extension kit from Promega according to the manufacturer's instructions. The product was analyzed on a 6% denaturing polyacrylamide gel.
Generation of Various Lengths of 5' Genomic Sequence/Luciferase Reporter Constructs-- Various lengths of 5' genomic fragments were prepared by restriction digestion of pDS, and then ligated into a luciferase reporter vector pGL3basic (Promega). Constructs A and B contain the longest insertion and were generated by digesting pDS with SmaI and NotI, filling protruding ends with Klenow, and inserting into the SmaI site of pGL3basic. The promoter direction is the same as that of the reporter gene in construct A and the opposite direction in construct B. Construct C was made by digestion of pDS with HindIII and insertion into the HindIII site of pGL3basic. The promoter direction is the same as that of the reporter construct. Construct D has the same insert as construct C but the opposite orientation. Construct E was prepared in two steps; pDS was digested with PstI, and the generated 1.15-kb fragment was inserted into pBluescript. The resulting plasmid was cut by BamHI and HindIII and then ligated into pGL3basic previously digested with BglII and HindIII. Construct F was made by digestion of pDS with EarI and EcoRV. The resulting 1-kb EarI-EcoRV fragment was purified and ligated into SmaI-digested pGL3basic. Construct G was prepared by EcoRI digestion of pDS followed by Klenow fill-in, and the 650-bp fragment was purified and inserted into SmaI-digested pGL3basic. Constructs H and I were derived from construct C: construct C was digested with XhoI and the 5.3-kb fragment was purified and self-ligated to generate construct H. Construct I was made in a similar way as construct H except KpnI was used instead of XhoI. Construct J was generated by EcoRI digestion of construct C, and the 5.9-kb fragment was purified and self-ligated. The identity and the orientation of the constructs were verified by multiple restriction mapping and partial sequencing.
Cell Culture-- COS-7 and RAW264.7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT). U937 and P388D1 cells were cultured in RPMI 1640 supplemented with 10% and 15% fetal bovine serum, respectively.
Transient Transfection and Reporter Activity
Assays--
Transfection in COS-7 and RAW264.7 cells was carried out
using LipofectAMINE (Life Technologies, Inc.). One µg of promoter construct DNA, and 1 µg of pSV--galactosidase control plasmid (Promega) were cotransfected into ~ 4 × 105
cells. After 48 h, the cells were harvested and the luciferase and
-galactosidase activities were determined with assay kits from
Promega and Tropix, Inc. (Bedford, MA), respectively. Transfection of
U937 and P388D1 cells was conducted by electroporation with a Bio-Rad
Gene Pulser at 300 V and 960 microfarads. Fifteen µg of promoter
construct and 10 µg of pSV-
-galactosidase DNA were cotransfected
into ~ 7 × 106 cells, which were harvested
after 17 h. The luciferase and
-galactosidase activity assays
were performed according to the manufacturer's instructions.
PAF Acetylhydrolase Enzymatic Assay-- Secreted PAF acetylhydrolase activity was assessed by collecting conditioned medium and then determined directly as described (23).
Reverse Transcriptase PCR-- Total RNA was isolated from macrophages using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH). The first strand of cDNA was synthesized from 1 µg of total RNA using Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). The PCR reaction was carried out using two primers, which amplify a 562-bp cDNA fragment in the PAF acetylhydrolase coding sequence. The intensity of the DNA bands was quantitated and normalized to the intensity of the glyceraldehyde-3-phosphate dehydrogenase bands. The quantitation was performed as described above.
Immunoblot with PAF Acetylhydrolase Antibody-- Macrophages were lysed with CHAPS lysis buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 16 mM CHAPS, 0.5 mM dithiothreitol, 1 mM benzamidine HCl, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor), and the protein concentration was determined by the BCA protein assay (Pierce). Fifty µg of protein were loaded on a 12% SDS-polyacrylamide gel. Western blots were carried out as described (24). A rabbit polyclonal plasma PAF acetylhydrolase antibody was used as the primary antibody. The secondary antibody was a horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Life Science). The immunodetection was performed using an ECL Western blotting detection system (Amersham). The intensity of the bands was quantified as described above.
Gel Mobility Shift Assays--
Nuclear extracts from monocytes
and macrophages were prepared as described previously (25). The
oligonucleotide probes were chemically synthesized and then were
annealed in STE buffer at 100 °C for 10 min and slowly cooled down
to room temperature. The annealed, double-stranded oligonucleotides
were separated from the single-stranded oligonucleotides on a 5% low
melt agarose gel. The double-stranded oligonucleotides were purified by
phenol extraction of the recovered fragment. The probes were labeled with [-32P]ATP using T4 polynucleotide kinase and then
separated from free [
-32P]ATP through a NucTrap probe
purification column. The binding reaction was performed using a gel
shift assay system from Promega. Nuclear extracts (5 µg) were
incubated with ~ 50,000 cpm of radioactive probe. The bound
products then were analyzed on a 6% native polyacrylamide gel.
Gels were dried and exposed to Kodak BioMax MR films (Eastman Kodak
Co.) overnight at
70 °C.
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RESULTS |
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Tissue Distribution of Plasma PAF Acetylhydrolase-- Our original studies using Northern blot analysis (11) did not detect mRNA for the plasma form of PAF acetylhydrolase in liver, which conflicted with studies that observed secretion of the activity by cultured hepatocytes (18, 22, 26). Therefore, we examined the mRNA expression with a more sensitive method and a more extensive sample of tissue mRNAs. This experiment (Fig. 1) revealed that there is widespread expression of the mRNA for PAF acetylhydrolase but that the levels differed by as much as 4-fold. The mRNA was expressed in all parts of the brain, but at low levels except for hippocampus, which had the highest expression of the brain tissues. The tissues with the highest levels of expression included ovary, placenta, liver, lymph node, and thyroid gland in adult, and the spleen in the fetus. The lung had a lower, but easily detectable, amount. Thus, this experiment confirmed most of the previously reported pattern but, significantly, identified the liver as a potential source. This widespread distribution is consistent with two interpretations; either many cell types express the mRNA for PAF acetylhydrolase, or one cell type that is found in many tissues expresses it. The latter seemed more probable because we had found previously that macrophages, which are in all tissues, secrete high levels of enzyme activity. However, endothelial cells also are found in most organs, but we found no mRNA and no secretion of the PAF acetylhydrolase activity from endothelial cells (data not shown). Thus, we conclude that the expression of PAF acetylhydrolase in many tissues is most likely the result of expression in tissue macrophages. In the studies reported here, we focused on the regulation of PAF acetylhydrolase expression in macrophages.
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Regulation of Plasma PAF Acetylhydrolase Expression by Inflammatory
Mediators--
Several cytokines have been shown to alter the levels
of PAF acetylhydrolase activity in vivo, and in some
in vitro studies. To determine the basis for this response,
we examined the PAF acetylhydrolase activity secreted by
monocyte-derived macrophages with or without cytokine treatment.
Compared with cells without treatment, cells that were treated with
IFN, IL1
, IL4, IL6, TNF
, GM-CSF (granulocyte/macrophage
colony-stimulating factor), M-CSF (macrophage colony-stimulating
factor), IFN
, and LPS all secreted less PAF acetylhydrolase
activity, although the effect was modest in most cases (data not
shown). We also measured the intracellular activity in the same samples
to ensure that the effect on the extracellular activity did not result
just from an inhibition of the secretory process; the intracellular
levels paralleled (but were more pronounced than) the changes observed in the supernatant, indicating that the effect of cytokines was to
decrease the synthesis of PAF acetylhydrolase (data not shown). IFN
and LPS caused the most marked inhibition on PAF acetylhydrolase secretion by 25% and 34%, respectively. These two cytokines have been
shown to synergistically stimulate the expression of the nitric oxide
synthase in a macrophage cell line (27). This prompted us to test the
effect of IFN
and LPS in combination. Macrophages were incubated
with IFN
, LPS, or IFN
plus LPS for 24 h, and the secreted
PAF acetylhydrolase activity was determined. The combined treatment
resulted in a 25% reduction of the secreted activity, which is the
same level as that observed using IFN
alone (data not shown).
Therefore, there was no synergistic or additive effect of IFN
and
LPS on PAF acetylhydrolase expression.
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Determination of the Transcriptional Initiation Site and Isolation and Characterization of the Promoter Region-- Primer extension was carried out to precisely map the transcriptional initiation site (+1 site). We designed an antisense oligonucleotide that started 40 bases downstream of the 5' end of the cDNA construct obtained from library screening (11), and performed the extension reaction with mRNA extracted from monocyte-derived macrophages. A single extension product of 107 bases was generated as determined by electrophoresis in a denaturing polyacrylamide gel (Fig. 3). Therefore, we conclude that the +1 site is 67 bases upstream of the 5' end of the originally identified cDNA. The position was further confirmed by primer extension using two additional antisense primers, both of which identified the same site (data not shown).
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Inflammatory Mediators Alter PAF Acetylhydrolase Expression by
Regulating Transcription--
For studies regarding the effects of
cytokines on PAF acetylhydrolase transcription, we used the mouse
macrophagic cell line RAW264.7 since it was much more readily
transfected than human monocyte-derived macrophages. This cell line is
an appropriate model since several previous investigators have reported
that this line expresses receptors for a variety of cytokines (30, 31),
and we found that treatment of these cells with murine cytokines
inhibited the synthesis of PAF acetylhydrolase as assessed by secreted
activity (data not shown). Construct A, which contains the entire 3.5 kb of 5' genomic sequence, was transiently transfected into cells
supplemented with various cytokines, and luciferase activity was
determined 16 h later. We found that IFN and LPS had a marked
inhibitory effect on PAF acetylhydrolase transcription (Fig.
6), which is in agreement with our
activity determinations. IFN
decreased the promoter activity by
about 35%, and LPS reduced it by 50%. Thus, the inhibitory effects of
IFN
and LPS on PAF acetylhydrolase levels can be explained at a
transcriptional regulation level.
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The Increased PAF Acetylhydrolase Expression during Macrophage Differentiation Is Transcriptionally Regulated-- We and others have shown that the synthesis and secretion of the plasma form of PAF acetylhydrolase is increased dramatically during differentiation of monocytes, or monocyte-like leukemic cell lines, to macrophages (11, 19). This response may be important in regulating the amount of pro-inflammatory lipids in tissues and could be a mechanism for resolving the acute phase of inflammation (14). To examine whether this response is regulated at the transcriptional level, we compared the relative activity of the PAF acetylhydrolase promoter in a monocytic cell (U937) to that in two macrophagic cell lines (RAW264.7 and P388D1). We found that the promoter activity in U937 cells was about 3-fold higher than that of the pGL3basic vector (a value about one-third lower than that observed in COS-7 cells). In contrast, the promoter activities in RAW264.7 and P388D1 cells were about 8- and 16-fold higher than that of the pGL3basic vector, respectively (Table I). This result indicates that the promoter activity is macrophage differentiation dependent, and implies that the remarkable difference of mRNA expression between monocytes and macrophages is likely the result of negative regulation of the promoter activity in monocytes and positive regulation in macrophages.
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DISCUSSION |
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In this study, we found that LPS and IFN, which activate many
of the functions of macrophages during inflammation, transcriptionally repress PAF acetylhydrolase expression. In contrast, PAF acting via its
receptor is a positive transcription regulator of this gene. In
addition, different levels of promoter activity were observed in
monocytic and macrophagic cells, strongly suggesting that the PAF
acetylhydrolase promoter is regulated in both positive and negative
ways during differentiation.
To perform these studies, we cloned and characterized the PAF
acetylhydrolase promoter and mapped the transcriptional initiation site
to an adenosine residue located 67 bases upstream of the 5' end of the
cDNA construct. The promoter does not contain a TATA box, which is
typically found upstream (25 to
30) of the transcriptional
initiation site of many eukaryotic genes. This type of promoter is
commonly found in housekeeping genes and in hematopoietic
lineage-specific genes (32, 33). Many of the TATA-less promoters share
a weak consensus sequence (initiator element, INr) around the
transcriptional initiation site, CA+1NT/AYY (34). The
sequence around the +1 site in the PAF acetylhydrolase promoter,
CA+1GCCC, is quite similar to the consensus INr sequence.
The presence of three Sp1 sites (GC boxes) at
203 and +18 in a short
distance from the initiation site implies that Sp1 transcription
factors contribute to the precise transcription initiation.
Sequence comparison among the promoter regions of many genes expressed by myeloid cells revealed three consensus motifs: CCCCACCC, CCCCTCCC, and CCCTTCC (35). The first two motifs have been demonstrated to bind to transcription factor MS2, which is expressed only by differentiated myeloid cells but not by undifferentiated counterparts (36, 37). The MS2 consensus sequence is almost always found in the promoters of myeloid-specific and developmentally controlled genes. The third motif probably binds to another myeloid-cell specific general transcription activator. In the PAF acetylhydrolase promoter, there are seven regions resembling the MS2 consensus sequence. In addition, the sequence from +207 to +217, GGGAGGAAGGG, contains overlapping binding sites for MS2 (GGGAGG) (36), Pu.1/MS1 transcription activator (GAGGAA), and MS1/MS2 (GAGGAAG). Pu.1 is a member of the Ets family of transcription activators that is only expressed in macrophages and B cells, and MS1 is a general transcription factor expressed exclusively in myelomonocytic cells (35, 36). The presence of binding sites for MS1 and Pu.1 in the PAF acetylhydrolase promoter strongly suggests that the plasma PAF acetylhydrolase gene is a myeloid-specific gene. Moreover, multiple potential binding sites for MS2 indicate that the expression of this gene is under tight differentiation control, which is likely to be primarily mediated by the MS2 transcription activator. The role of MS2 in the regulation of expression of the PAF acetylhydrolase gene is currently under investigation. Finally, tissues known to contain large numbers of macrophages such as ovary, placenta, liver, lymph node, thyroid gland, and fetal spleen also express higher levels of PAF acetylhydrolase mRNA, which is in agreement with the idea that expression of the PAF acetylhydrolase gene is specific for mature myeloid cells.
In addition to MS2, other differentiation-induced transcription factors
may be involved in the regulation of expression of the PAF
acetylhydrolase gene. A well characterized macrophage-specific transcription factor is the zinc finger protein Egr-1, which has been
shown to be essential for differentiation of myeloblast cells along the
macrophage lineage (38). No region in the 1.3-kb 5' genomic sequence
perfectly matches the consensus sequence GCGGGGGCG for Egr-1 binding.
However, there is a sequence from +208 to +216, CCGGGGGCG, which
differs in only one nucleotide from the consensus motif. Whether Egr-1
binds to this region and is involved in the regulation of expression of
the PAF acetylhydrolase gene awaits further investigation. Recently,
there have been reports that differentiation of myeloid cells is
accompanied by the activation of another transcription factor, DIF
(differentiation-induced factor) (39, 40). In U937 cells, DIF was shown
to be activated by phorbol esters, IFN, GM-CSF, and CSF-1. This
myeloid differentiation-related transcription factor was further shown
to be a STAT5-related transcription factor that binds to a STAT
consensus sequence (41). Our data indicate that nuclear factors from
monocytes and macrophages bind to regions containing STAT consensus
sequences in a differentiation-dependent manner (Fig. 8).
Therefore, it is possible that the STAT5-related DIF is involved in the
differentiation-dependent regulation of the PAF
acetylhydrolase promoter by interacting with regions containing the
STAT consensus sequences, i.e.
692 to
712 and
631 to
656. Since nuclear factors from both monocytes and macrophages bind to these two regions, we anticipate that the regulatory mechanism in
these two regions involves negative regulation in monocytes and
positive regulation in macrophages. Our observation on the different
levels of promoter activity in monocytes and macrophages (Table I)
supports this possibility.
We found that PAF stimulates the expression of its own inactivating enzyme. PAF has been observed to increase the expression of other genes related to inflammation and injury. Bazan and colleagues reported that PAF rapidly activated transcription of the proto-oncogene c-fos in neuroblastoma and corneal epithelial cells, and the responsive cis region was mapped to an 80-bp promoter region (42). They also found that human immunodeficiency virus long terminal repeat promoter activity was elevated by PAF (5). In addition, PAF induced collagen type I transcription in corneal tissues, and PAF and retinoic acid synergistically induced the expression of prostaglandin synthase II (43, 44). Our data suggest that PAF functions as a physiological feedback regulator of its catabolic enzyme: an elevated level of PAF in plasma or tissues leads to enhanced PAF acetylhydrolase expression and secretion. A recent in vivo study using a rat model observed a rapid increase in PAF receptor expression in the ileum after PAF injection (45). Taken together with our data, it is likely that PAF can precisely modulate cellular responses, and therefore probably the entire body can adjust the capacity and magnitude of inflammatory and immune responses partly by this autoregulatory mechanism.
Previous evidence indicates that most of the physiological actions of
PAF can be achieved at very low concentrations (109 to
10
12 M) (2). In the promoter-reporter system
used, we did not see a stimulatory effect of PAF until the
concentration reached 10 nM (10
8
M) (Fig. 7). Several possibilities can explain the
requirement of higher PAF concentrations in our experiments. Specific
plasma or membrane factors may facilitate access of PAF to its receptor in vivo, and these factors may be absent in our experimental
approach (i.e. cultured cells utilized, culture medium used,
etc.). Alternatively, our experimental conditions may have hindered
ready access of PAF to the promoter (for example sequestration by
albumin or other proteins present in the system could account for the
higher EC50). In addition, the sensitivity of the assay may
have limited our ability to detect small alterations in promoter
activity at lower PAF levels. In related studies, other research groups
also have shown that higher PAF concentrations in vitro are
necessary to elicit biological effects that are characteristic of the
actions of PAF in vivo (6, 42).
In our studies, the effect of PAF was mediated specifically through its
receptor since the stimulation was completely blocked by pretreatment
of cells with PAF antagonists (Fig. 7). The PAF receptor is a
G-protein-coupled molecule that can activate several signal
transduction pathways, including phospholipases C, D, and A2, protein kinase C, and tyrosine kinases (46-48).
Although PAF regulates the expression of a number of genes, the nature
of the activated transcription factors and the
cis-responsive elements have not been characterized yet. One
study showed that PAF induced NFB activation through a
G-protein-coupled pathway (49), but the PAF acetylhydrolase promoter
does not have a perfect match for the NF
B consensus sequence.
LPS and IFN were found to inhibit PAF acetylhydrolase promoter
activity in our studies. IFN
exerts important biological functions
in the immune response including activation of macrophages, promotion
of differentiation, and antiviral action (50). It sometimes functions
synergistically with other stimuli such as LPS (endotoxin), which is a
cell wall constituent of Gram-negative organisms that can trigger
inflammatory reactions during infections. The effect of IFN
is known
to be mediated through the JAK-STAT pathway, which targets the STAT
consensus sequence (also termed GAS elements) (28). The effect of LPS
was demonstrated to be mediated in part by IL1
, IL1
, and TNF
receptors in decidual macrophages (20). Moreover, the effect of LPS has
been shown to involve several transcription factors including NF
B
and NFIL6 (51). Recently, one study reported that LPS induced the
binding of NF
B components NF
B1 and RelA to the STAT consensus
sequence (31). A significant synergism has been observed between IFN
and LPS on transcription of nitric oxide synthase (27) in the macrophage cell line RAW264.7, which was also used in our study. The
regulation of the PAF acetylhydrolase gene had a different pattern, in
that IFN
and LPS did not show a synergistic, or even additive,
effect. This probably ruled out the possibility that IFN
and LPS
bind to two cis-regulatory elements, and that the two
regions either interact with each other (synergistic) or function independently (additive) during the regulation. Therefore, IFN
and
LPS probably share the same responsive cis-region in the PAF acetylhydrolase promoter. Regardless of the precise mechanism, both
stimuli lowered PAF acetylhydrolase expression and secretion in our
study, suggesting that they may potentiate the inflammatory response by
this mechanism.
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ACKNOWLEDGEMENTS |
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We are grateful to Donelle Benson and Deborah Dykstra for their excellent technical assistance in monocyte isolation and culture. We thank Elie Traer and Diana Lim for their expert assistance on computer analysis of sequencing data and figure preparation. We also thank the DNA Sequencing and Peptide/Oligo DNA Synthesis Core Facilities at the University of Utah (supported by Grant CA42014 from the NCI, National Institutes of Health). We thank Dr. James Ihle for generously providing purified STAT proteins.
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Note added in proof |
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While this manuscript was under review, the in vivo effect of LPS on PAF acetylhydrolase was reported in Kupffer cells (52).
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL 50153 and HL 35828 (to S. M. P.) and National Institutes of Health Postdoctoral Fellowship 1F32HL09775 (to Y. C.).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. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF027357.
¶ To whom correspondence should be addressed: Huntsman Cancer Institute, 15 N. 2030 E., Suite 4220, University of Utah, Salt Lake City, UT 84112-5330. Tel.: 801-585-3401; Fax: 801-585-6345; E-mail: steve.prescott{at}genetics.utah.edu.
1
The abbreviations used are: PAF,
platelet-activating factor; LPS, lipopolysaccharide; kb, kilobase(s);
IFN, interferon-
; IL, interleukin; bp, base pair(s); TNF
,
tumor necrosis factor-
; PCR, polymerase chain reaction; STAT, signal
transducer and activator of transcription; GM-CSF,
granulocyte/macrophage colony-stimulating factor; M-CSF, macrophage
colony-stimulating factor; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
TPA, 12-O-tetradecanoylphorbol-13-acetate; DIF,
differentiation-induced factor.
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