From the Departments of Biochemistry and
§ Medicine, Queen's University, Kingston, Ontario K7L 3N6,
Canada
Received for publication, September 18, 2002, and in revised form, December 20, 2002
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ABSTRACT |
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Thrombin-activable fibrinolysis inhibitor (TAFI)
has recently been identified as a positive acute phase protein in mice,
an observation that may have important implications for the interaction of the coagulation, fibrinolytic, and inflammatory systems. Activated TAFI (TAFIa) inhibits fibrinolysis by removing the carboxyl-terminal lysines from partially degraded fibrin that are important for maximally
efficient plasminogen activation. In addition, TAFIa has been shown to
be capable of removing the carboxyl-terminal arginine residues from the
anaphylatoxins and bradykinin, thus implying a role for the TAFI
pathway in the vascular responses to inflammation. In the current
study, we investigated the ability of acute phase mediators to modulate
human TAFI gene expression in cultured human hepatoma (HepG2) cells.
Surprisingly, we found that treatment of HepG2 cells with a combination
of interleukin (IL)-1 Thrombin activable fibrinolysis inhibitor
(TAFI)1 was first identified
in 1989 by two independent groups as a basic carboxypeptidase present
in fresh serum that was distinct from the constitutive basic
carboxypeptidase N (1, 2). By virtue of the intrinsic instability of
this enzyme, whose activity disappeared within 2 h upon incubation
at 37 °C, Hendriks et al. (1) designated the novel
activity "unstable" carboxypeptidase or carboxypeptidase U (1).
Campbell and Okada (2) determined that the enzyme removed arginine
residues from substrates more efficiently than lysines and therefore
designated it carboxypeptidase R (2). In 1991, Eaton et al.
(3) isolated a cDNA encoding the zymogen form of the enzyme and
found that it was highly homologous to pancreatic
procarboxypeptidase B. Bajzar et al. (4)
independently isolated a protein on the basis of its ability to inhibit
fibrinolysis in the setting of sustained activation of the coagulation
cascade; on the basis of this property, they named the protein TAFI.
Amino acid sequence analysis of TAFI revealed it to be identical to plasma procarboxypeptidase B and procarboxypeptidases U and R. TAFI can
be activated by thrombin (4), plasmin (5), and thrombin in complex with
thrombomodulin (6), with the last being by far the most efficient
activator. Activated TAFI (TAFIa) inhibits fibrin clot lysis by
removing the carboxyl-terminal lysine residues from partially degraded
fibrin that mediate positive feedback in the fibrinolytic cascade (7).
As such, it has been hypothesized that TAFI plays a role in
vivo in mediating the balance between coagulation and fibrinolysis.
Additional substrates for TAFIa have been identified that imply a role
for the TAFI pathway beyond inhibition of fibrinolysis. TAFIa has been
shown to remove the carboxyl-terminal arginines from the anaphylatoxin
peptides C3a and C5a (8) as well as from bradykinin (9-11). As such,
TAFIa may modulate inflammatory processes in the vasculature in the
setting of activation of the coagulation cascade. Additional evidence
for a role for the TAFI pathway in inflammation comes from the recent
observation that TAFI is an acute phase protein in mice; injection of
the animals with bacterial lipopolysaccharide (LPS) elicited increases
in both plasma TAFI concentrations and hepatic TAFI mRNA abundance (12). In order to begin to assess if human TAFI is also an acute phase
protein and to determine the molecular mechanisms underlying this
potential phenomenon, we have studied the ability of acute phase
mediators to alter TAFI gene expression in a cultured human hepatoma cell model.
Materials--
Restriction and modification enzymes were from
New England Biolabs, Invitrogen, Promega, and Stratagene.
[ Cell Culture and RNA Analysis--
HepG2 cells (human
hepatocellular carcinoma) (13) were grown in MEM containing 10% fetal
calf serum and 1% PSF. Cells were maintained in a humidified 37 °C
incubator under a 95% air, 5% CO2 atmosphere. Cytokine(s)
and/or dexamethasone were added to the growth medium, and the cells
were incubated for 24 h prior to harvesting the RNA from the
cells. In some experiments, after a 24-h treatment with cytokine(s),
actinomycin C1 was added to the cultures (to 5 µg/ml) and
incubation continued (in the presence of cytokine(s), where
appropriate) for different times up to 8 h prior to harvesting the
RNA. RNA was isolated using TRIzol reagent as recommended by the
manufacturer. Poly(A)+ RNA was prepared using the
NucleoTrap mRNA purification kit. For Northern blot analysis, total
RNA (20 µg/lane) or poly(A)+ RNA (~2 µg/lane) (in
50% (v/v) formamide, 10 mM MOPS, pH 7.0, 2.2 M
formaldehyde) was incubated at 65 °C for 15 min, quenched on ice,
and then fractionated on a 1% (w/v) agarose gel containing 10 mM MOPS, pH 7.0, 2.2 M formaldehyde. The RNA
was blotted onto a nylon membrane (Hybond-XL, Amersham Biosciences) by
capillary transfer in 20× SSC (1× SSC is 15 mM trisodium
citrate, pH 7, 150 mM NaCl). After ultraviolet
cross-linking of the RNA to the membrane, blots were hybridized with
radiolabeled probes corresponding to the TAFI cDNA (full open
reading frame) (3) or the Reporter Plasmids--
The luciferase reporter plasmids
TAFI[-2699]-luc, TAFI[-1128]-luc, TAFI[-236]-luc, and
TAFI[-73]-luc have been described previously (18). The plasmids
contain fragments of the 5'-flanking region of the human TAFI gene
inserted into the luciferase reporter vector pGL3 Basic (Promega). The
numbering refers to the 5'-most nucleotide of genomic DNA included in
the construct. The 3'-boundary of all the reporter plasmids is a
HindIII restriction site immediately downstream of the
initiator methionine codon such that all possible sites for
transcription initiation are present in the construct; the initiator
methionine codon was mutated to TTG in all cases. Additional
luciferase reporter plasmids representing progressive 5' deletions
between
A mutant variant of TAFI[-1128]-luc (designated
TAFI[-1128/ Expression Plasmids--
Glucocorticoid receptor (GR) expression
plasmids were the generous gift of Dr. Robert Haché (University
of Ottawa, Ottawa, Canada). pTL2-GR consists of a 2.9-kb
BamHI restriction fragment encompassing the full-length open
reading frame of the rat GR (20) inserted into pTL2 (a derivative of
pSG5, Ref. 21, containing an expanded multiple cloning site) digested
with BglII and BamHI. GAL0-540C encodes a fusion
protein containing the Saccharomyces cerevisiae GAL4
DNA-binding domain fused to a carboxyl-terminal fragment of the rat GR
(amino acids 540-795; encompasses the ligand binding domain).
rGR-L501P is a mutant variant of the full-length rat GR in which amino
acid 501 has been changed from a leucine to a proline. rGR(N525)-pTL2
(encoding the amino-terminal 525 amino acids of the rat GR) was
constructed as follows: pEGFP-GRwtN525 (pEGFP-C1
(Clontech) containing a rat GR fragment
encompassing amino acids 22-525) was digested with MluI
(downstream of the simian virus 40 (SV40) polyadenylation site), the
ends were made blunt with T4 DNA polymerase, and the plasmid was then
digested with BglII (within the rat GR coding sequence). The
resultant fragment was inserted into rGR-pTL2 in which the
corresponding sequences of the wild-type receptor cDNA were removed
by digestion with BamHI (at which point the ends were made
blunt) and BglII. Expression of all rat GR variants in
mammalian cells was driven by the SV40 early promoter with the
exception of rGR-L501P, whose expression was driven by the Rous sarcoma
virus (RSV) long terminal repeat.
Reporter Gene Assays--
For luciferase reporter gene assays,
HepG2 cells were grown in 6-well plates (Corning) and transfected by
the method of calcium phosphate co-precipitation (22). Typically, cells
received ~1.3 µg of luciferase reporter plasmid and 0.6 µg of
Gel Mobility Shift Assays--
Complementary sets of
oligonucleotides encompassing the putative GRE in the TAFI promoter
were synthesized: sense 5'-CAC AGG AAC AAG AGG GAA CAT GCC GTT ATA TTT
TAA CC-3'; antisense 5'-GGT TAA AAT ATA ACG GCA TGT TCC CTC TTG TTC CTG
TG-3'. Mutant oligonucleotides encompassing the same range,
corresponding to the mutation in the GRE (see above), were also
synthesized. For radiolabeled TAFI binding site probes for gel mobility
shift assays, 5 pmol of sense strand oligonucleotide was end-labeled
using [
Binding reactions were performed in binding buffer (20 mM
HEPES pH 7.9, 60 mM KCl, 5 mM
MgCl2, 5 µg/ml bovine serum albumin, 10% (v/v) glycerol,
2 mM dithiothreitol) and contained 1 µl of partially
purified recombinant human GR expressed in insect cells as well as 2.0 µg of poly(dIdC)·(dIdC) and 10 fmol of radiolabeled probe
(~20,000 cpm). Binding reactions were incubated for 30 min on ice.
Some binding reactions contained a 10-, 50-, 100-, or 200-fold molar
excess of unlabeled binding site competitor (corresponding either to
the wild-type TAFI sequence, the sequence containing the mutant GRE, or
the TAT-GRE). Reactions were loaded on a 6% polyacrylamide gel in
0.5× Tris borate-EDTA, 5% (v/v) glycerol that had been
pre-electrophoresed at 300 V for 1 h at 4 °C. Electrophoresis was continued for a further 2.5 h at this temperature, at which time the gel was fixed, dried, and exposed to film (Kodak BIOMAX MR).
Effect of Acute Phase Mediators on TAFI Gene Expression--
It
has been demonstrated that injection of mice with bacterial
lipopolysaccharide (LPS) results in an increase in both plasma TAFI
concentrations as well as hepatic TAFI mRNA abundance (12). To
determine which mediators of the acute phase response regulate TAFI
gene expression, we treated human hepatoma (HepG2) cells for 24 h
with the cytokines interleukin (IL)-1
To verify that our cell culture model was a valid model for the
analysis of the effect of acute phase mediators on TAFI gene expression, we measured the abundance of the mRNA for the Effect of Acute Phase Mediators on TAFI Promoter
Activity--
To examine whether the acute phase mediators were
capable of specifically altering the activity of the TAFI promoter,
HepG2 cells were transiently transfected with a luciferase reporter plasmid containing the 5'-flanking region of the human TAFI gene. The
transfected cells were treated for 24 h with the acute phase mediators described above, and luciferase activity was quantitated in
extracts isolated from the cells as a measure of TAFI promoter activity
(Fig. 2). Dexamethasone (1.0 µM) stimulated TAFI promoter activity more than 2-fold,
either alone or in combination with IL-6. None of the other treatments
had an appreciable effect on TAFI promoter activity.
Effect of Acute Phase Mediators on TAFI mRNA
Stability--
Since the ability of IL-1 Identification of a Glucocorticoid Response Element in the TAFI
Promoter--
In order to identify sequences in the TAFI promoter that
mediate the increase in promoter activity elicited by dexamethasone, we
utilized a series of luciferase reporter plasmids containing progressive 5' deletions of 5'-flanking sequence (Fig.
4A). The plasmids were
transiently transfected into HepG2 cells, and the cells were incubated
either in the absence or presence of 1.0 µM dexamethasone
for 42 h prior to harvest and luciferase assay. Full
responsiveness to dexamethasone was preserved upon deletion of
5'-flanking sequence up to nucleotide
Glucocorticoid hormones exert their effects on transcription through
binding to and activating the GR, a member of the nuclear receptor
superfamily (29). Ligand-bound GR binds as a homodimer to specific
sequences, known as GREs, in the promoters of target genes
thereby stimulating their transcription. Inspection of the sequence
downstream of
In order to examine the requirement for DNA binding of the GR for
dexamethasone-dependent activation of the TAFI promoter, we
performed co-transfection experiments with the wild-type or
While ectopic expression of the intact, full-length rGR substantially
increased the magnitude of dexamethasone induction of the wild-type
TAFI promoter (Fig. 6B), ectopic expression of the rGR
variants lacking the ability to bind DNA had little or no influence on
the magnitude of induction. Expression of the N525 variant resulted in
a large, dexamethasone-independent induction of the TAFI promoter. This
result was not unexpected given that removal of the ligand-binding
domain results in a constitutively active GR. Interestingly, while
ectopic expression of the intact, full-length rGR did not mediate any
dexamethasone-dependent induction of the
TAFI[-1128/
To demonstrate explicitly that the GR can bind to the TAFI promoter
GRE, gel mobility shift assays were performed using radiolabeled double-stranded oligonucleotide probes corresponding to the wild-type TAFI GRE and recombinant human GR expressed in insect cells (Fig. 7A). As a positive control,
gel mobility shift assays were also performed using the GRE from the
TAT gene (Fig. 7B). The autoradiograms show a
complex of low mobility is formed using the probe containing the
wild-type TAFI-GRE as well as the TAT-GRE. These complexes represent
specific binding of the GR because they are competed effectively by an
excess of unlabeled oligonucleotides containing the TAT-GRE and the
wild-type TAFI-GRE but not effectively by the mutant TAFI-GRE
(TAFI- The acute phase reaction is a complex host defense mechanism that,
in response to triggers such as trauma, surgery, tissue infarction, or
severe infection, aims to counteract the underlying challenge while
restoring homeostasis (reviewed in Refs. 31 and 32). Among the features
of the acute phase response are systemic changes such as fever,
increases in neutrophil production, changes in lipid and amino acid
metabolism and activation of the coagulation and complement cascades as
well as changes in the expression of a panel of liver-expressed plasma
proteins. These acute-phase proteins are classified as either positive
or negative acute phase proteins depending on whether their expression
is induced or repressed, respectively, in the acute phase. Among the
acute phase proteins are C-reactive protein, serum amyloid A,
Regulation of the expression of acute phase proteins is most often at
the level of their transcription in liver and is largely a function of
the action of certain inflammatory cytokines (reviewed in Ref. 33).
Acute phase proteins are divided into two broad categories based on the
cytokines that regulate their expression: class I proteins, including
serum amyloid A, C-reactive protein, complement factor C3, and
A study in which mice were injected intraperitoneally with a lethal
dose of bacterial lipopolysaccharide, a maneuver that would be expected
to provoke a robust acute phase response, resulted in increases in
concentrations of TAFI in plasma as well as in hepatic TAFI mRNA
abundance (12). This study, therefore, identified TAFI as a positive
acute phase protein. A role for the TAFI pathway in the acute phase
response is reasonable to expect since this pathway may impact both on
hemostatic as well as inflammatory functions. Activation of TAFI, with
the attendant inhibition of fibrinolysis (4, 7), may stabilize clots
formed in response to tissue damage or as a means to isolate regions of
severe infection. On the other hand, the TAFI pathway may influence the
vascular responses to inflammatory stress: the ability of TAFIa to
remove the carboxyl-terminal arginine residues from C3a and C5a (the anaphylatoxins) and from bradykinin could have effects on vascular tone
and permeability (8-11).
The potential increase in plasma TAFI concentrations in the acute phase
may reflect a requirement for enhanced activity of the TAFI pathway
during host defense. Alternatively, the activation of the coagulation
and fibrinolytic systems that occurs during the acute phase may result
in consumption of the existing pool of plasma TAFI that could be
compensated for by an increase in hepatic TAFI expression. Further
analysis of the TAFI pathway during the acute phase will likely yield
valuable insights into the role of this pathway in regulating the
balance between coagulation and fibrinolysis and in mediating
interactions between the coagulation and inflammatory systems.
We have utilized a cultured human hepatoma cell model to assess if TAFI
gene transcription is induced by acute phase mediators and to
investigate the molecular mechanisms underlying these effects. HepG2
cells represent a well characterized model system for the study of
transcriptional regulation in the acute phase, as they retain many of
the characteristics of hepatocytes, endogenously express many
liver-specific genes, and contain cell surface receptors for the
relevant inflammatory cytokines (13, 33). Surprisingly, we found that
treatment of HepG2 cells with IL-1 We identified a functional glucocorticoid response element in the TAFI
promoter between In the study in mice that identified TAFI as a positive acute phase
protein (12), the magnitude of the increase in hepatic TAFI mRNA
abundance was not carefully measured; as such, it is difficult to
directly compare it to our in vitro data. Nonetheless, it is
perhaps surprising that the acute phase mediators we examined either
increased TAFI gene expression by a relatively modest amount (~2-fold
increase mediated by dexamethasone) or even decreased TAFI gene
expression (~60% decrease mediated by IL-1 No data currently exist that explicitly address potential changes in
TAFI plasma concentrations in the acute phase in humans, although
associations between TAFI concentrations and those of fibrinogen (38,
39)2 and C-reactive
protein2 (38) have been reported, suggesting that TAFI gene
expression could be positively regulated by inflammatory mediators.
Intracellular signals elicited by IL-1 ultimately result in the
activation of certain transcription factors, specifically AP-1,
NF- The association of TAFI concentrations with inflammatory markers and
fibrinogen suggests a role for glucocorticoid hormones (or other
inflammatory mediators) in regulating plasma TAFI levels in the setting
of chronic inflammation. For example, one study found that compared
with preoperative plasma TAFI levels in patients requiring coronary
artery bypass grafting (which were higher than in healthy controls),
postoperative plasma TAFI levels fell 17% by day 3, then rose again
14% by day 6 (38). On the other hand, glucocorticoid hormones are
important clinically as anti-inflammatory drugs. Interestingly, one
study compared the distribution of plasma TAFI concentrations in a
sample of patients with rheumatoid arthritis with that in a healthy
control population: plasma TAFI concentrations are clearly higher in
patients with rheumatoid arthritis, although the use of glucocorticoids
in the patients was not accounted for (36).
In conclusion, we have documented the effect of acute phase mediators
on endogenous TAFI gene expression in HepG2 cells by Northern blot
analysis as well as on TAFI promoter activity by transient transfection
into HepG2 cells of luciferase reporter plasmids harboring the TAFI
5'-flanking region. We found that when administered in combination,
IL-1 and IL-6 suppressed endogenous TAFI mRNA
abundance in HepG2 cells (~60% decrease), while treatment with
IL-1
or IL-6 alone had no effect. Treatment with IL-1
and/or IL-6
had no effect on TAFI promoter activity as measured using a luciferase
reporter plasmid containing the human TAFI 5'-flanking region,
whereas treatment with IL-1
and IL-6 in combination, but not alone,
decreased the stability of the endogenous TAFI mRNA. Treatment with
the synthetic glucocorticoid dexamethasone resulted in a 2-fold
increase of both TAFI mRNA levels and promoter activity. We
identified a functional glucocorticoid response element (GRE) in the
human TAFI promoter between nucleotides
92 and
78. The GRE
was capable of binding the glucocorticoid receptor, as assessed by gel
mobility shift assays, and mutation of this element markedly decreased the ability of the TAFI promoter to be activated by dexamethasone.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP, [
-32P]dATP, and
poly(dIdC)·(dIdC) was purchased from Amersham Biosciences. TRIzol
reagent, minimum essential medium (MEM), Dulbecco's modified Eagle's
medium/nutrient mixture F-12 (DMEM/F-12), and
penicillin-streptomycin-Fungizone (PSF) were obtained from Invitrogen.
Fetal calf serum was purchased from ICN. NucleoTrap mRNA
purification kits were from Clontech. Synthetic
oligonucleotides were purchased from Cortec DNA Service Laboratories,
Inc. (Kingston, Ontario, Canada). IL-1
, IL-6,
dexamethasone, and actinomycin C1 were purchased from Sigma
and were reconstituted as recommended by the manufacturer. Recombinant
human glucocorticoid receptor expressed in insect cells and a
double-stranded oligonucleotide corresponding to the human tyrosine
aminotransferase glucocorticoid response element (TAT-GRE) was obtained
from Affinity Bioreagents, Inc. A cDNA clone corresponding to the
full-length mRNA encoding the human
-chain of fibrinogen was
obtained from the American Type Culture Collection.
-chain of human fibrinogen (14). In order
to correct for differences in RNA loading and transfer, blots were
stripped and hybridized with radiolabeled probes corresponding to
either the 36B4 cDNA (human acidic ribosomal phosphoprotein PO)
(15) or, in the case of RNA from cells treated with actinomycin
C1, the glyceraldehyde-6-phosphate dehydrogenase cDNA;
in the latter experiments, the amount of TAFI RNA present at each time
point was calculated as described by Wilson and Deeley (16), assuming a
half-life for the glyceraldehyde-6-phosphate dehydrogenase mRNA of
8 h (17). Probes were prepared using [
-32P]dATP
and the Prime-It II random primer labeling kit (Stratagene). Hybridization was carried out at 68 °C for 1 h in ExpressHyb
solution (Clontech). The blots were washed at room
temperature in 1× SSC, 0.1% (w/v) SDS, and then at 50 °C in 0.2×
SSC, 0.1% (w/v) SDS. Blots were exposed to a storage phosphor screen
(Kodak), and band intensities were quantitated using a Molecular Imager
FX phosphorimager (BioRad).
236 and
73 (TAFI[-120]-luc, TAFI[-100]-luc, TAFI[-90]-luc, and TAFI[-80]-luc) were constructed using PCR in which the 5'-most nucleotide of the upstream primer corresponds to the
indicated nucleotide in the name of the reporter plasmid.
GRE]-luc) was constructed using PCR according to the
method of Nelson and Long (19). The sequence of the mutagenic
oligonucleotide was as follows:
5'-CACAGGAACAAGAGGGACAGTGCCGTTATATTTTAACC-3'; the
underlined nucleotides (positions
89 to
87 of the TAFI promoter) are mismatches relative to the wild-type sequence.
-galactosidase internal control plasmid (RSV-
gal) (23) (to
control for transfection and harvesting efficiency). In some
experiments, cells also received 0.6 µg of GR expression plasmids.
After a 6-h exposure to the precipitate, the cells were washed three
times in phosphate-buffered saline and given fresh medium, and
incubation was continued for an additional 42 h. In some
experiments, hormones or cytokines were added to the culture medium at
various times during the 42-h incubation period. The cells were
harvested for preparation of cytoplasmic extracts for luciferase and
-galactosidase assays as previously described (18). For each sample,
the relative luciferase activity was calculated to be the luciferase
activity per unit of
-galactosidase activity per unit volume of cell extract.
-32P]ATP and T4 polynucleotide kinase.
Unincorporated label was removed using a NAP-5 column (Amersham
Biosciences). The labeled oligonucleotide was combined with a 5-fold
molar excess of cold antisense oligonucleotide, and the two annealed by
placing in boiling water and allowing to cool slowly at room
temperature. Unlabeled TAFI competitor binding site probes were made by
annealing equimolar amounts of sense and antisense oligonucleotides.
For the TAT-GRE (sense: 5'-CTA GGC TGT ACA GGA TGT TCT GCC TAG-3';
antisense: 5'-CTA GGC AGA ACA TCC TGT ACA GCC TAG-3'), 5 pmol of the
double-stranded oligonucleotide was labeled as described above, and
then unincorporated label was removed using a NAP-5 column. Unlabeled
competitor binding site probes were diluted from the stock TAT-GRE
provided by the manufacturer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and/or -6, as well as
dexamethasone (a synthetic glucocorticoid hormone analog). We measured
the abundance of the endogenous TAFI mRNA by Northern blot
analysis, using the acid ribosomal phosphoprotein PO (36B4) mRNA as
an internal standard (Fig. 1). While we
found that TAFI mRNA abundance was actually decreased by treatment
of the cells with a combination of IL-1
and IL-6, IL-1
alone or
IL-6 either alone or in combination with dexamethasone had little or no
effect. Treatment with dexamethasone resulted in
dose-dependent changes in TAFI mRNA abundance: an
increase of up to 2-fold was observed that peaked at doses between 0.5 and 2 µM hormone.
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Fig. 1.
Effect of acute phase mediators on TAFI gene
expression in HepG2 cells. HepG2 cells were treated with the
indicated combinations of IL-1 (1 ng/ml), IL-6 (10 ng/ml), and
dexamethasone (dex) (0.2-5.0 µM) for 24 h. Total RNA was harvested from the cells and poly(A)+ RNA
was prepared. Northern blot analysis was performed on 1-2 µg of
poly(A)+ RNA or 20 µg of total RNA. Blots were hybridized
to a radiolabeled cDNA probe encompassing the entire TAFI open
reading frame (left panel) or the full-length
-chain of
human fibrinogen (
-Fgn; right panel); to correct for
differences in RNA loading and transfer, the blots were stripped and
hybridized to a radiolabeled cDNA probe corresponding to the acid
ribosomal phosphoprotein PO (36B4). Band intensities were quantified
using a phosphorimager; shown are the corrected intensities of the TAFI
or
-Fgn bands, with mRNA abundance under each condition
presented relative to that in the absence of acute phase mediators
(control). The data shown are the mean ± S.E. from
three independent blots.
-chain of fibrinogen (
-Fgn) under similar conditions. As has been
previously reported (24), both IL-6 and dexamethasone induce expression of
-Fgn, with the greatest effect occurring when the two mediators are administered in combination. The magnitude of the induction by IL-6
plus dexamethasone was, however, less than has been reported for the
mRNA levels of rat
-Fgn in primary rat hepatocytes (15-20-fold) (24) or the activity of the rat
-Fgn promoter in H35 rat
hepatoma cells (10-fold) (25). This finding may result from the fact that HepG2 cells are relatively deficient in glucocorticoid receptor (26). In keeping with published reports (27), IL-1
had the effect of
blunting the induction of
-Fgn mRNA stimulated by IL-6 and IL-6
plus dexamethasone.
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Fig. 2.
Effect of acute phase mediators on TAFI
promoter activity in HepG2 cells. HepG2 cells were transiently
transfected, by the method of calcium phosphate co-precipitation, with
a luciferase reporter plasmid (TAFI[-2699]-luc) containing the
5'-flanking region of the human TAFI gene (up to 2699 bp upstream of
one of the transcription start sites). Also included in each
transfection was the internal control plasmid RSV- gal to correct for
differences in transfection and harvesting efficiency. After a 6-h
exposure to the precipitate, the cells were washed and provided with
fresh medium containing the indicated combinations of IL-1
(1 ng/ml), IL-6 (10 ng/ml), and dexamethasone (dex) (1.0 µM). After a further 42-h incubation, cytoplasmic
extracts were prepared for the measurement of luciferase activity as
well as
-galactosidase activity to allow for correction for
differences in transfection and harvesting efficiency. Relative
luciferase activities (mean ± S.E. of three independent
experiments) are defined as luciferase activity per unit of
-galactosidase activity and are shown as a percentage of that
observed in the absence of acute phase mediators
(control).
and IL-6 in combination to
decrease TAFI mRNA abundance did not seem to result from a decrease in TAFI promoter activity, we investigated whether these cytokines could influence the stability of the TAFI mRNA transcript. HepG2 cells were treated for 24 h with IL-1
and/or IL-6 at which time transcription was arrested by the addition of actinomycin
C1. RNA was harvested at different times after the addition
of actinomycin C1 and the remaining TAFI mRNA
quantified by Northern blot analysis (Fig.
3). The results show that the TAFI
mRNA has an intrinsic half-life of about 3 h, identifying the
TAFI transcript as a relatively short-lived mRNA species (28);
addition of IL-1
and IL-6 in combination results in a
destabilization of the TAFI mRNA whereas either cytokine
administered alone had no effect. From regression of the Northern blot
data, the effect of the combined cytokines results in a 22% decrease
in the half-life of the TAFI transcript.
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Fig. 3.
IL-1 and IL-6
decrease the stability of the TAFI mRNA. HepG2 cells were
treated with IL-1
(1 ng/ml) and/or IL-6 (10 ng/ml) for 24 h.
Actinomycin C1 was added to 5 µg/ml and incubation was
continued in the presence of cytokine(s). RNA was harvested at
different times after the addition of actinomycin C1 and
the abundance of remaining TAFI mRNA quantitated by Northern blot
analysis. Differences in RNA loading and transfer were accounted for by
using GAPDH mRNA as an internal standard. The abundance of TAFI
mRNA after actinomycin C1 addition is shown relative to
that present immediately before the addition of actinomycin
C1. The data shown are the mean ± S.E. of three
independent experiments.
100 (the numbering refers to
the number of nucleotides upstream of one of the transcription start
sites in the TAFI promoter) (Fig. 4B). However, deletion of
sequences between
100 and
90 resulted in almost complete elimination of the increase in promoter activity in response to dexamethasone. Deletion of sequences up to
80 does not result in a
further decrease in dexamethasone responsiveness, while deletion of
sequences up to
73 results in a complete loss of TAFI promoter activity, as we have previously reported (18).
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Fig. 4.
Localization of a GRE in the human TAFI
promoter. Panel A, a series of luciferase
(luc) reporter plasmids was prepared that contain
progressive 5' deletions of the 5'-flanking region of the human TAFI
gene (open boxes). The numbering refers to the 5'-most
nucleotide of the 5'-flanking region in each construct, and the
bent arrow refers to the location of the transcription start
sites. Panel B, HepG2 cells were transiently transfected
with the luciferase reporter plasmids shown in panel A. Also
included in each transfection was the internal control plasmid
RSV- gal to correct for differences in transfection and harvesting
efficiency. After a 6-h transfection period, the cells were washed and
incubated for a further 42 h in the absence (filled
bars) or presence (open bars) of 1.0 µM
dexamethasone (dex), at which time cytoplasmic extracts were
prepared for the measurement of luciferase and
-galactosidase
activities. Relative luciferase activities are defined as luciferase
activity per unit of
-galactosidase activity and are shown as a
percentage of that for the TAFI[-2699]-luc reporter plasmid in the
absence of dexamethasone. The data shown are the mean ± S.E. of
triplicate transfections; similar results were observed in two
independent experiments.
100 of the TAFI promoter revealed the presence of a
sequence, between
92 and
78, that resembles a consensus GRE (30) in
that it is an imperfect inverted repeat with a 3-nucleotide spacing of
the 6-nucleotide half-sites (Fig. 5A). Mutations were introduced
into this candidate GRE that would be expected to abolish its ability
to bind GR (Fig. 5A). HepG2 cells were transfected with a
luciferase reporter plasmid encompassing the mutations and including
the TAFI 5'-flanking sequence up to
1128
(TAFI[-1128/
GRE]-luc), or the corresponding wild-type
reporter plasmid (TAFI[-1128]-luc), and the cells were treated with
dexamethasone (Fig. 5B). The mutations greatly decreased the
response of the TAFI promoter; interestingly, the decrease in the
extent of the response was similar to that observed upon deletion of
sequences between
100 and
90.
View larger version (20K):
[in a new window]
Fig. 5.
Mutagenesis of the GRE in the human TAFI
promoter. Panel A, the consensus GRE is an imperfect
inverted repeat of 6-nucleotide half-sites with a 3-nucleotide spacing
(30); the underlined nucleotides in the consensus are
present in >80% of GREs (30). The TAFI promoter contains an imperfect
inverted repeat resembling this consensus between 92 and
78. Also
shown is the GRE from the rat
1-acid glycoprotein gene,
which contains a stretch of ten consecutive nucleotides that are
identical in the TAFI gene. A mutation of the putative TAFI promoter
GRE (
GRE), indicated below the TAFI promoter sequence,
was introduced into the luciferase reporter plasmid
TAFI[-1128]-luc to create the plasmid TAFI[-1128/
GRE]-luc.
Panel B, HepG2 cells were transiently transfected with the
luciferase reporter plasmids TAFI[-1128]-luc and
TAFI[-1128/
GRE]-luc. Also included in each transfection was the
internal control plasmid RSV-
gal to correct for differences in
transfection and harvesting efficiency. After a 6-h transfection
period, the cells were washed and incubated for a further 42 h in
the absence (filled bars) or presence (open bars)
of 1.0 µM dexamethasone (dex), at which time
cytoplasmic extracts were prepared for the measurement of luciferase
and
-galactosidase activities. Relative luciferase activities are
defined as luciferase activity per unit of
-galactosidase activity.
The data shown are the mean ± S.E. of triplicate transfections;
similar results were observed in two independent experiments.
GRE
mutant TAFI promoter reporter plasmid and expression plasmids for rat
GR (rGR) variants. The variants used (see Fig.
6A) included the full-length
wild-type receptor (rGR-wt), a variant including only the
amino-terminal 525 amino acids (i.e. lacking the DNA- and
ligand-binding domains; rGR-N525), a variant including only the
carboxyl-terminal 540 amino acids fused to the GAL4 DNA binding domain
(i.e. lacking the amino-terminal transactivation domain and
the DNA binding domain of the GR; rGR-540C), and a mutant of the
full-length GR with a single point mutation that abolishes DNA binding
(rGR-L501P).
View larger version (19K):
[in a new window]
Fig. 6.
Effect of ectopic expression of GR variants
on dexamethasone induction of the TAFI promoter. Panel
A, schematic representation of the rat GR variants used. The
top line represents the topology of the rat GR. Indicated is
the numbering of the amino acids as well as the locations of the
DNA-binding (DNA) and glucocorticoid-binding
(ligand) domains. rGR-wt is the full-length wild-type GR.
rGR-N525 corresponds to the amino-terminal 525 amino acids. rGR-540C is
a fusion protein consisting of the GAL4 DNA-binding domain
(DBD) fused to the carboxyl-terminal 256 amino acids of the
rat GR. rGR-L501P is a mutant variant of the full-length rat GR
containing a leucine to proline substitution at amino acid position
501. Panel B, HepG2 cells were transiently transfected with
the luciferase reporter plasmids TAFI[-1128]-luc or
TAFI[-1128/ GRE]-luc. Some transfections also included expression
plasmids for the GR variants described in panel A. Each
transfection included the internal control plasmid RSV-
gal to
correct for differences in transfection and harvesting efficiency.
After a 6-h transfection period, the cells were washed and incubated
for a further 42 h in the absence (filled bars) or
presence (open bars) of 1.0 µM dexamethasone
(dex), at which time cytoplasmic extracts were prepared for
the measurement of luciferase and
-galactosidase activities.
Relative luciferase activities are defined as luciferase activity per
unit
-galactosidase activity. The data shown are the mean ± S.E. of three independent experiments.
GRE]-luc reporter plasmid, the two rGR variants lacking
DNA-binding ability were able to confer an ~2-fold induction by this
hormone. The constitutively active N525 rGR variant induced this mutant
reporter plasmid in a hormone-independent fashion, albeit to a
substantially reduced extent compared with the wild-type TAFI[-1128]-luc plasmid.
GRE). No specific complexes were observed when radiolabeled
oligonucleotides containing the mutant TAFI GRE were utilized (data not
shown).
View larger version (40K):
[in a new window]
Fig. 7.
Binding of GR to the TAFI
promoter GRE. End-labeled, double-stranded binding site probes
were prepared corresponding to the wild-type TAFI promoter GRE
(TAFI-GRE) (panel A) or the GRE contained in the
TAT promoter (TAT-GRE) (panel B).
Probes were incubated with or without partially purified recombinant
full-length human GR; some binding reactions contained increasing
amounts (a 10-, 50-, 100-, or 200-fold molar excess), as indicated, of
unlabeled competitor binding site probes corresponding to the TAT-GRE,
the TAFI-GRE, or the mutant GRE described in the legend to Fig. 5
(TAFI- GRE). Binding reactions were subjected to
electrophoresis on a non-denaturing polyacrylamide gel, and bands were
detected by autoradiography. Indicated to the left of the
autoradiograms are the locations of migration of the unbound probe
(F) as well as a complex of low mobility (B) that
binds specifically to the functional TAT and TAFI GREs. NS
denotes a nonspecific complex detected using the TAFI-GRE probe.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-acid glycoprotein, components of the complement
cascade, proteins of the coagulation and fibrinolytic cascades,
protease inhibitors, and proteins involved in transport and
inflammatory functions. The magnitude of the changes in expression can
be small (such as a 50% increase in the expression of ceruloplasmin)
to vast (such as a greater than 1000-fold increase in the expression of
C-reactive protein and serum amyloid A).
1-acid glycoprotein, are induced by IL-1-type cytokines
in concert with IL-6-type cytokines; class II proteins, including
fibrinogen, haptoglobin, and
2-macroglobulin, are
induced by IL-6-type cytokines with the participation of glucocorticoid hormones.
and IL-6 in combination reduced
expression of the endogenous TAFI gene, as assessed by Northern blot
analysis (Fig. 1). However, we did not observe an effect of this
combination of cytokines on TAFI promoter activity measured by
transient transfection of luciferase reporter plasmids (Fig. 2).
Indeed, we found that these two cytokines, when administered in
combination but not alone, were capable of decreasing the stability of
the TAFI mRNA, which likely accounts for the ability of these
cytokines to decrease TAFI mRNA abundance. We hypothesize that
IL-1
and IL-6 together modulate the expression of a factor or
factors that influences TAFI mRNA decay. IL-6 or IL-1
alone had
no effect on TAFI mRNA abundance in HepG2 cells or TAFI promoter
activity (Figs. 1 and 2). However, dexamethasone treatment, either in
the presence or absence of IL-6, increased TAFI promoter activity
~2-fold; dexamethasone alone increased TAFI mRNA abundance almost
2-fold but had no effect in the presence of IL-6.
92 and
78; mutation of this GRE greatly decreased
the ability of the TAFI promoter to be activated by dexamethasone (Fig.
5B) and of the TAFI-GRE to bind to the GR (Fig. 7). Of note,
the unlabeled TAFI-GRE was a less effective competitor for binding of
GR to either probe than unlabeled positive control TAT-GRE (Fig. 7),
suggesting that the latter GRE possesses a higher affinity for GR.
Indeed, the TAFI GRE contains numerous key substitutions in the
downstream half-site sequence (Fig. 5A), relative to the
consensus GRE (30) and the TAT-GRE, which differs from the consensus at
only one position in the upstream half-site (TGTACAGGATGTTCT). In addition, a small but detectable
extent of competition by the unlabeled TAFI-
GRE was observed with
both the TAFI-GRE and TAT-GRE probes (Fig. 7), suggesting that the TAFI-
GRE retains a weak affinity for the GR. Indeed, the TAFI promoter containing the mutant GRE retains a small extent of
dexamethasone inducibility (Fig. 5B) and exhibits
dexamethasone-independent induction by ectopic expression of the
constitutively active N525 GR variant, albeit to a reduced extent
relative to the wild-type promoter (Fig. 6B). On the other
hand, ectopically expressed full-length GR abolishes the small extent
of dexamethasone inducibility of the mutant TAFI promoter (Fig.
6B). An explanation for this finding might be that the
overexpressed full-length receptor consumed factors required for
dexamethasone-dependent transactivation of the mutant TAFI
promoter. That the TAFI-GRE is not an optimal GR binding sequence is
perhaps consistent with the relatively modest extent to which
dexamethasone induces transcription of the TAFI promoter, compared with
the TAT promoter (e.g. 8-10-fold increase in TAT
mRNA in primary rat hepatocytes induced by 1 µM dexamethasone, Ref. 34).
+ IL-6). However, it
is important to stress that even small changes in plasma concentrations
of TAFI can be expected to impact on the potential of the TAFI pathway
to influence fibrinolysis. The range of plasma concentrations of TAFI
(the upper limit of which has been reported to exceed 200-400
nM; Refs. 35-37) is well below the Km
for activation of TAFI by thrombin or thrombin-thrombomodulin (~1
µM; Ref. 6); as such, a change in concentration of TAFI in plasma would result in a corresponding change in the rate of TAFI
activation by thrombin or thrombin-thrombomodulin. Indeed, a strong
correlation between plasma TAFI concentrations and in vitro
clot lysis times has been observed (35, 37).
B, and C/EBP
(33). IL-6 signaling results in the activation of
the transcription factor STAT3 (33). Functional cooperation between GR
and all of these transcription factors has been reported (40-44). In
addition to the functional GRE, we have identified a functional C/EBP
binding site between
52 and
40 of the TAFI promoter (45).
Examination of the TAFI gene 5'-flanking sequence for consensus
transcription factor binding sites using Matinspector (46) revealed
potential binding sites for AP-1 but not STAT3 or NF-
B (data not
shown). Alternatively, it cannot be ruled out that in humans, TAFI is either not an acute phase protein or is a negative acute phase protein.
Different acute phase proteins have distinct temporal patterns of
induction and subsequent return to baseline (31); the temporal pattern
of TAFI expression during the acute phase could reflect the changing
balance between cytokine and glucocorticoid signaling pathways over time.
and IL-6 decreased TAFI mRNA abundance by 60%, an effect
that was associated with a destabilization of the TAFI mRNA
transcript. Dexamethasone resulted in a 2-fold increase in both TAFI
mRNA abundance and promoter activity, and we were able to identify
a functional GRE in the TAFI promoter. Further studies will be required
to fully elucidate the significance of these observations with respect
to regulation of TAFI gene expression in the acute phase and other
inflammatory states.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Robert Haché (University of Ottawa, Ottawa, Canada) for the gift of the glucocorticoid receptor expression plasmids.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant MOP-36491 from the Canadian Institutes for Health Research.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.
¶ To whom correspondence should be addressed: Dept. of Biochemistry, Room A208 Botterell Hall, Queen's University, Kingston, Ontario K7L 3N6, Canada. Tel.: 613-533-6586; Fax: 613-533-2987; E-mail: mk11@post.queensu.ca.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M209588200
2 P. Crainich, Z. Tang, E. M. Macy, M. B. Boffa, M. E. Nesheim, M. L. Koschinsky, and R. P. Tracy, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: TAFI, thrombin-activable fibrinolysis inhibitor; C/EBP, CCAAT/enhancer-binding protein; GR, glucocorticoid receptor; rGR, rat GR; GRE, glucocorticoid response element, IL, interleukin; PSF, penicillin-streptomycin-Fungizone; TAFIa, activated TAFI; TAT, tyrosine aminotransferase; MOPS, 4-morpholinepropanesulfonic acid.
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