© 1997 American Heart Association, Inc.
Articles |
Intracellular Calcium Mobilization Suppresses the TNF-
–Stimulated Synthesis of PAI-1 in Human Endothelial Cells
Indications That Calcium Acts at a Translational Level
From INSERM CJF 93-12, Laboratoire d'Hématologie, Faculté de Médecine, Marseille, France.
Correspondence to Gilles Nalbone, INSERM CJF 93-12, Laboratoire d'Hématologie, Faculté de Médecine, 27 Bd Jean Moulin, 13385 Marseille, Cedex 05, France.
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Abstract |
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Abstract We investigated in human umbilical vein endothelial cells (HUVECs) the interaction between the signaling pathways triggered by calcium mobilization and those affected by human recombinant tumor necrosis factor-
(TNF) on the expression
of type-1 plasminogen activator
inhibitor (PAI-1). Calcium ionophore A23187 alone exerted a
modest increase (50%) on PAI-1 synthesis. TNF alone increased PAI-1
accumulation in the culture medium in a time- and dose-dependent
fashion, but this increase was abolished when A23187 was added
simultaneously with TNF. The downregulating effect of
A23187 was not the result of impaired protein secretion, proteolysis,
cytotoxicity, or an apoptotic process. A23187 did not decrease
the TNF-enhanced PAI-1 mRNA level but did provoke a significant shift
in the distribution pattern of PAI-1 transcripts by increasing the
2.3-kb relative to the 3.2-kb form. Comparable inhibitory
effects on PAI-1 protein synthesis were observed when A23187 was added
7 hours after the onset of TNF stimulation, strongly suggesting a
posttranscriptional inhibitory action of calcium signaling
on TNF-stimulated PAI-1 synthesis. However, treatment with actinomycin
D showed that PAI-1 mRNA stability was not altered by the various
treatments. Chelation of extracellular calcium by EGTA did not prevent
the A23187-induced inhibition of TNF-stimulated PAI-1 protein
synthesis, emphasizing the role of internal calcium stores in the
inhibition of PAI-1 synthesis. Sucrose gradient fractionation of cell
lysates revealed that regardless of which treatment was used, both
PAI-1 mRNA transcripts exhibited similar sedimentation profiles in the
actively translating polysomal pool, suggesting that the A23187-induced
shift had no functional consequence on translation. However, in
TNF-stimulated cells, A23187 induced a higher proportion of PAI-1 mRNAs
that sedimented in fractions corresponding to less dense polysomes, a
phenomenon that usually reflects a slower initiation rate during mRNA
translation. A23187 also abolished the increase in PAI-1 synthesis
induced by recombinant human interleukin 1ß, and thapsigargin exerted
effects comparable to those of A23187 on PAI-1 synthesis in
TNF-stimulated cells. It is proposed that in HUVECs, the A23187-induced
release of calcium from endoplasmic stores suppresses at the
translational level the increase in PAI-1 synthesis triggered by
proinflammatory cytokines.
Key Words: endothelium • PAI-1 • cytokines • calcium • translation
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Introduction |
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The endothelium has a critical role in the regulation of fibrinolysis.1 A reduction in endothelial fibrinolytic potential is thought to contribute to fibrin accumulation and therefore to thrombosis, which is the most frequent complication in the advanced stages of atherosclerosis. In this event, PAI-1, the main physiological inhibitor of TPA and urokinase, has a key function. High plasma levels of PAI-1 are found in patients with ischemic heart diseases such as angina pectoris and recurrent myocardial infarction and in patients with the metabolic syndrome of insulin resistance.2 3
Besides its implication in fibrin accumulation in the intravascular and extravascular space, PAI-1 is also thought to be involved in the control of local extracellular matrix degradation in the vessel wall.4 5 The fibrinolytic cascade and more specifically PAI-1 are regulating components of this process, as suggested by results from in vitro6 and in situ7 8 9 10 observations. This notion is reinforced by in vivo experiments with transgenic mice lacking a functional PAI-1 gene and subjected to arterial trauma. These mice rapidly develop intimal cell proliferation and thickening when compared with control animals.11
In atherosclerotic lesions, ECs are exposed to various
simultaneous stimuli and are constrained to integrate them.
Consequently, the transduction process of an agonist may be
significantly altered when it acts under these
pathophysiological conditions. The action of
proinflammatory cytokines is an example of a transduction
pathway that can be modulated by other stimuli, such as
calcium-mobilizing effectors. TNF-–activated transduction
processes such as protein kinase, free radical, or lipid messenger
pathways, may cross-talk in vivo with those of oxidized
LDL,12 shear stress,13 14 or hormones that
bind to G protein–coupled receptors that trigger intracellular calcium
mobilization. Interestingly, calcium-mobilizing agonists were
demonstrated to alter the specific effect of TNF on protein expression
by either augmenting or suppressing it. For example, TNF and bradykinin
when acting together potentiate the calcium signal in human tracheal
smooth muscle cells.15 We have shown that in the human
promonocytic cell line U937, TNF activated PAI-1 synthesis, a
process that was strongly potentiated by the calcium ionophore A23187
or thapsigargin.16 Calcium-mobilizing agents can suppress
the TNF-induced expression of vascular cell adhesion molecule-1 in
murine brain microvascular endothelial
cells,17 the inducible nitric oxyde synthase in human
chondrocytes,18 and the gelatinase activities in HT-1080
cells.19 The mechanism of the regulatory action of calcium
on TNF signaling has not been identified. It could be related to some
interacting roles of kinases, such as mitogen-activated protein
kinases20 21 22 23 24 or calcium/calmodulin–dependent
protein kinases25 26 because these are known to be
activated by TNF and calcium mobilization. Although TNF is
known to enhance PAI-1 expression in various cell types, including
ECs,27 28 little is known of the interactions between
calcium and cytokine signaling on PAI-1 synthesis in these
cells.
Therefore, we investigated in human ECs the effects of TNF, with or without a calcium-mobilizing agent, on PAI-1 synthesis at the levels of mRNA and protein syntheses. Results show that calcium mobilization itself moderately stimulates PAI-1 synthesis but drastically inhibits the TNF-induced increase in PAI-1 synthesis. This depressing effect is not due to a general process of cytotoxicity or apoptosis, nor to a lower transcription rate or mRNA stability. It could be related to an impaired translation process of the PAI-1 mRNA.
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Chemicals
FCS and cell-culture media were purchased from Eurobio. Calcium ionophore A23187, TG, DMSO, cycloheximide, actinomycin D, DTT, sucrose, and Triton X-100 were purchased from Sigma Chemical Co. Human recombinant TNF-
(specific activity, 3.8x107 U/mg) was
from Euromedex, and human recombinant IL-1ß (5x107
U/mg), CDP-Star, and anti–digoxigenin-AP (Fab fragments) were from
Boehringer Mannheim. Monoclonal antibodies (15H12, 12A4, and
7D4 and 15H12 coupled to protein A–Sepharose) and cDNA probes specific
for human PAI-1 and human GAPDH cDNA probes were a gift from the Center
for Thrombosis and Vascular Research, Leuven, Belgium. The ELISA kit
specific for human PAI-1 antigen (Asserachrom PAI-1) was from Stago.
Positively charged nylon membranes and agarose were from
Appligène. Fluo 3-AM was purchased from Molecular Probes, Inc.
[3H]Leucine and [35S]methionine were
from DuPont-NEN.
Cell Culture
ECs were isolated from human umbilical cord veins according to
the method of Jaffe et al29 and were cultured into
25-cm2 tissue-culture flasks coated with 10 mg/mL
calf skin gelatin. Cells were grown to confluence at 37°C in Ham's
F12/Eagle's MEM (vol/vol) supplemented with 20%
heat-inactivated FCS, 2 mmol/L
L-glutamine, 100 IU/mL penicillin, 100 µg/mL
streptomycin, 50 IU/mL heparin, and 37.5 µg/mL EC growth
supplement under a 5% CO2 atmosphere. All experiments were
performed with once-passaged cultures that, unless indicated otherwise,
were performed in six-well culture plates containing 2 mL per well of
EM. The EM composition was the same as that listed above, except it
contained 5% instead of 20% FCS.
Cell Treatment
Essentially two kinds of stimulation protocol were used (Fig 1
). In protocol 1, TNF and A23187 were
added simultaneously. Thirty minutes later, the conditioned
EM was eliminated and the cell monolayer washed with fresh EM. Then TNF
only was added in a single dose to the EM and left for the indicated
times. In protocol 2, TNF and A23187 were added separately. Cells were
stimulated first by TNF for 7 hours and then treated for 30 minutes
with A23187. The conditioned EM was eliminated and the cell monolayer
washed with fresh EM. Then the cells were incubated in EM without TNF
for the indicated times. In each protocol, stimulation by TNF or A23187
alone was also performed under the same conditions of washing and
incubation as described above. In the experiments performed with
IL-1ß or TG, protocol 1 was applied. Cells without any treatment
(control cells) were included. A23187 and TG were dissolved in pure
ethanol and DMSO, respectively. Proper controls with an equivalent
volume (which never exceeded 0.25% vol/vol) of ethanol or DMSO
alone were proved not to affect the responses studied.
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Assays
Cell viability was assessed by measuring the activity of lactate
dehydrogenase that was released into the conditioned EM by using a
routine clinical assay performed on a automatic analyzer
(Hitachi 717, Boehringer Mannheim). The PAI-1 antigen assay was
performed on supernatants from conditioned EM or cell lysates by ELISA
as described by Declerck et al.30 In some experiments, we
also assayed antigenic PAI-1 with the commercial Asserachrom ELISA kit.
Results were expressed in nanograms of PAI-1 antigen per
103 cells.
Pulse-Chase Experiments
Global proteolysis was investigated according to a method
derived from that of Nicotera et al.31 Preconfluent cells
were labeled with [3H]leucine (0.5 µCi/mL) for 24
hours. After the cell monolayer was washed, the labeled confluent cells
were incubated with the EM for 5 hours and then treated. At the end of
the experiment (18 hours), the conditioned EM was saved and the cell
monolayer detached by trypsinization and centrifuged. The
resulting pellet was mixed with 0.5 mL of 3% ice-cold perchloric acid
and left for 20 minutes at 4°C, and the mixture was
centrifuged at 2500g for 10 minutes. The supernatant
(S1) was saved and the pellet (P1) solubilized
in 0.5 mL NaOH 1 mol/L and 1% SDS. The conditioned EM was
treated by perchloric acid under the same conditions as above, which
yielded another pellet (P2) and supernatant
(S2). The radioactivity (R) of the fractions was counted.
The proteolytic index was calculated as:
(RS1+RS2)/(RS1+RS2+RP1+RP2).
An increase in the proteolytic index reflects a higher rate of
intracellular proteolysis.
Intracellular-specific PAI-1 proteolysis was also investigated. Cells were stimulated according to protocol 2. Cells were first stimulated by TNF for 6 hours in EM. Then the EM was replaced by a serum- and methionine-free culture medium for 20 minutes, and 25 µCi/mL of [35S]methionine was added and left for 20 minutes (pulse). The monolayer was carefully washed twice with EM, and the chase was performed by incubating the cells for 15 minutes with EM containing a large excess of nonradioactive methionine (125 mg/L). After another wash, A23187 treatment was performed for 30 minutes, and the cells were subsequently incubated in EM. TNF was always present until the end of A23187 treatment. Cells were trypsinized at the indicated times and lysed in a 50 mmol/L Tris-HCl buffer (pH 8.0) containing 1% Triton X-100 and Pefabloc SC, which is a mixture of protease inhibitors. After centrifugation, the supernatant was added to 25 µL of a mixture of PAI-1 monoclonal antibodies (15H12 and 7D4) coupled to protein A–Sepharose and analyzed as previously described.32 After electrophoresis, the gel was treated with the amplifier Amplify (Amersham), dried, and exposed to autoradiographic film for 2 weeks.
Protein Synthesis
The rate of global protein synthesis was assessed by measuring
[3H]leucine incorporation into total proteins obtained
from the perchloric acid–insoluble fraction of the cells. To this end,
[3H]leucine (0.5 µCi/mL) was added to the EM 10 hours
after stimulation and left for an additional 2 hours. The monolayer was
detached by trypsinization, and the perchloric acid–insoluble
fractions were obtained and counted as described above. Results were
expressed as disintegrations per minute per 104 cells.
Apoptosis
The apoptotic process was investigated by analyzing DNA
fragmentation after agarose gel electrophoresis. In brief, HUVECs
cultured in 25-cm2 flasks were trypsinized and then lysed
in PBS–0.1% Triton X-100 buffer for 20 minutes at 4°C. The
supernatant was incubated for 6 hours at 50°C with proteinase K (100
µg/mL) and then with RNase (50 µg/mL) for 2 hours at
37°C. The precipitated material was resuspended in Tris-EDTA buffer
and subjected to agarose gel electrophoresis (1.25%) containing
ethidium bromide.
Preparation of RNA, cRNA Probes, and Northern Blots
PAI-1 and GAPDH antisense cRNA probes were transcribed in
vitro from linearized recombinant plasmids (containing the 436-bp
fragment from nucleotides 1045 to 1481 of human PAI-1 cDNA
and the 359-bp fragment from nucleotides 20 to 379 of human
GAPDH cDNA) by using digoxigenin-11-UTP and SP6 RNA polymerase
(Boehringer Mannheim) according to the protocol described by
Melton et al.33 Total RNA of HUVECs was extracted
according to the method of Chomczynski and Sacchi,34 and 5
µg was analyzed by Northern blotting as previously
described.16 Detection of the respective mRNA-cRNA hybrids
was performed using a chemiluminescent detection kit as described by
the manufacturer (Boehringer Mannheim Biochemical No. 1363514).
The membrane was exposed to autoradiographic film for a
period of time ranging between 5 and 20 minutes to fit the
densitometric linear range. The levels of PAI-1 mRNAs were quantified
by densitometric analysis of the autoradiographic
films by using National Institutes of Health Image 1.54 software. To
normalize for variability in sample loading, PAI-1 mRNA signal density
values were expressed relative to GAPDH mRNA signal density.
Polysome Sucrose Gradients
Analysis of the distribution of polysome-associated
PAI-1 mRNAs was performed by centrifugation of
cytoplasmic extracts on a 15% to 50% sucrose gradient according to
the protocol described by Chen et al.35 A cytoplasmic
extract was obtained from HUVECs cultured in 150-cm2
flasks. The sucrose gradient was centrifuged at 40 000 rpm for
2 hours at 4°C in a Sorvall TH-641 swinging-bucket rotor. The
gradient was pumped from the bottom of the bucket, the optical density
at 254 nm recorded, and fractions of 
0.8 mL collected in sterile
tubes. Total RNA was immediately extracted and PAI-1 and GAPDH mRNAs
analyzed as described above.
Fluorescence Digital Imaging
The fluorescent probe Fluo 3-AM has been shown to be a
reliable dye to detect changes in cytosolic calcium
concentration.36 Cells were cultured on sterile glass
coverslips. Fluo 3-AM was dispersed in DMSO containing 20% (wt/vol)
pluronic acid. In all experiments, cells were incubated with 1
µmol/L Fluo 3-AM for 30 minutes, and experiments on dye-loaded
cells were done within the next 30 to 45 minutes. Analysis of
cell fluorescence was performed as previously
described.16 Changes in fluorescence were
recorded every 10 seconds for 3 to 4 minutes. Normalized
fluorescence in a single responsive cell was expressed as the
ratio of fluorescence at the indicated times after calcium
ionophore stimulation to the basal fluorescence just before
stimulation.
Statistical Analyses
Statistical significance of mean differences was investigated by
ANOVA and the Scheffé multiple-comparison method.
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Results |
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A23187 Inhibits TNF-Induced PAI-1 Accumulation in Conditioned EM
In these experiments protocol 1 was used. Incubation of HUVECs for 20 hours with increasing doses of TNF augmented the secretion of PAI-1 in the conditioned EM in a dose-dependent manner up to a concentration of 100 U/mL. At this dose, augmentation attained a factor of 3.3 when compared with PAI-1 secreted from control cells (Fig 2
). Therefore, in all subsequent studies,
the concentration of TNF was fixed at 100 U/mL. As shown in Fig 2, the
TNF-induced increase in PAI-1 accumulation in the conditioned EM became
measurable 10 hours after the beginning of stimulation and gradually
and significantly increased up to 20 hours. Treatment of cells with
cycloheximide (5 µmol/L) before TNF stimulation resulted
in PAI-1 levels below those of control cells (data not shown),
indicating that TNF did not induce a release of a preformed pool of
PAI-1. When HUVECs were treated with various concentrations of A23187
up to 10 µmol/L for 30 minutes, a moderate effect occured
at 5 µmol/L for which a 50% increase in PAI-1
accumulation was measured at 20 hours (data not shown). However, when
TNF and A23187 were added simultaneously, a dramatic
reduction of the TNF-induced increase in PAI-1 secreted into the
conditioned EM was observed (Fig 3). At
20 hours, a marked significant inhibition of TNF-induced PAI-1
accumulation was observed for doses of A23187 of 5 µmol/L
and 10 µmol/L, as the level of PAI-1 was close to that of
control cells. Time-dependent analysis showed that this
inhibitory effect became measurable 10 hours after
simultaneous addition of TNF and A23187 and was more marked
when measured for longer times of accumulation (Fig 3). The decrease in
antigenic PAI-1 in the conditioned EM was not attributable to a loss of
epitope recognition, because another pair of monoclonal antibodies
directed against other epitopes of PAI-1 gave the same results.
Therefore, we analyzed the possibility of impaired secretion,
intracellular proteolysis, an apoptotic process, or a defect in
protein synthesis.
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In TNF-Stimulated Cells, A23187 Did Not Impair PAI-1
Secretion
We analyzed the time-dependent evolution of intracellular
levels of PAI-1 according to protocol 1. As shown in Fig 4, TNF alone (100 U/mL) induced a higher
level of intracellular PAI-1 than in control cells. In TNF-stimulated
cells, A23187 (5 µmol/L) significantly reduced the
TNF-induced intracellular level of PAI-1. This indicates that in
TNF-stimulated cells, calcium mobilization induced by A23187 did not
provoke intracellular retention of PAI-1, which therefore was normally
secreted into the culture medium.
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In TNF-Stimulated Cells, A23187 Did Not Induce Proteolysis or
Apoptosis
As shown in Table 1, lactate
dehydrogenase release was not significantly altered by the various
treatments. A slight tendency to higher lactate dehydrogenase levels
was noticed when cells were treated simultaneously with TNF
and A23187. Assessment of intracellular proteolysis was investigated in
cells treated according to protocol 1. The proteolytic index,
determined as indicated in "Methods," was not significantly altered
by the various treatments (Table 1). To unmask a possible specific
PAI-1 proteolysis, we analyzed the fate of intracellular PAI-1
by pulse-chase experiments. Cells stimulated by TNF alone were compared
with those stimulated by both TNF and 7 hours later by A23187 (Fig 5) according to protocol 2, which was
more suited to this purpose. PAI-1 appeared mainly as the expected
50-kDa form, although some high-molecular-weight complexed
forms37 can be detected. The intensity of PAI-1 signals in
cells stimulated by TNF alone progressively decreased in a
time-dependent manner, likely reflecting secretion of the protein into
the EM. In cells treated with TNF and A23187, the time-dependent
pattern of PAI-1 signals was quite similar to that of cells treated
with TNF alone, strongly indicating that addition of A23187 did not
induce specific degradation of the protein.
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Many cell types treated with high doses of TNF or calcium-mobilizing
agents for a long time are well known to undergo apoptotic
processes (reviewed in Reference 3838 ). As shown in Fig 6, agarose gel electrophoresis of DNA
recovered in cell lysates failed to show any typical apoptotic
"ladder" pattern, suggesting that none of the treatments had
induced apoptosis. To further confirm that cells treated with
TNF and A23187 were not under sublethal conditions at 20 hours, at that
time we replaced the conditioned EM with fresh EM, again stimulated
HUVECs with TNF alone (100 U/mL), and measured the PAI-1 that
accumulated in the conditioned EM for an additional 18 hours. Results
were compared with those for control HUVECs incubated in EM for 20
hours and then stimulated for an additional 18 hours with TNF alone.
The TNF+A23187–pretreated HUVECs were able to augment PAI-1 synthesis
in response to a second TNF stimulation at levels (10.3±0.8
ng/103 cells) similar to those of control cells
(9.6±0.7 ng/103 cells). Therefore, inhibition by
A23187 of TNF-stimulated PAI-1 synthesis is a reversible process.
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In TNF-Stimulated Cells, A23187 Did Not Significantly Alter Global
Protein Synthesis
We then examined whether the decrease in PAI-1 synthesis was a
reflection of a drastic decrease in global protein synthesis. TNF alone
(100 U/mL) did not alter protein turnover (Table 1
). When associated
with A23187 (5 µmol/L), global protein turnover tended to
be lower (-16%) than in control or TNF-treated cells, although this
figure was not statistically significant. This indicates that in
TNF-stimulated cells, A23187 did not induce a drastic decrease in
protein synthesis. This prompted us to further investigate the action
of A23187 at the level of PAI-1 gene expression.
In TNF-Stimulated Cells, A23187 Did Not Alter the Level of
PAI-1 mRNAs
In human ECs, the PAI-1 gene is transcribed as two alternatively
polyadenylated mRNA transcripts (3.2 kb and 2.3 kb) differing
only by the lengths of their 3' untranslated region.39 40
Total RNA (5 µg) from control, TNF-, A23187-, and
TNF+A23187–stimulated cells was recovered at different times after the
beginning of stimulation according to protocol 1. The levels of PAI-1
mRNA in control cells did not change significantly throughout the
duration of the experiment (Fig 7A).
A23187 alone (5 µmol/L) exerted a slight transient
increase in PAI-1 mRNA at 6 to 7 hours (Fig 7A). The TNF-dependent
increase in PAI-1 mRNA levels in HUVECs has already been
described27 28 41 42 and has been demonstrated to be the
result of an enhanced transcriptional rate.42 In line with
these results, PAI-1 mRNA levels were markedly increased 5 hours after
TNF addition (100 U/mL) and attained a plateau at 7 hours that
persisted for several hours thereafter (Fig 7A). In cells stimulated
simultaneously with TNF (100 U/mL) and A23187 (5
µmol/L), PAI-1 mRNAs were present at levels comparable to
those of cells stimulated with TNF alone (Fig 7A). A peculiar feature
that can be noticed is the altered distribution of PAI-1 mRNAs after
A23187 treatment. In TNF-stimulated cells, examination of PAI-1 mRNA
distribution revealed that at 7 hours, the level of the 3.2-kb form
represented 72% of PAI-1 mRNAs (Fig 7B). However, 7 hours
after simultaneous addition of TNF and A23187, the 3.2-kb
transcript represented 45% of PAI-1 mRNA. The distribution
tended to recover its initial value after 18 hours.
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In TNF-Stimulated Cells, the Inhibitory Effect of
A23187 on PAI-1 Synthesis Is Posttranscriptional and Linked to
Internal Calcium Stores
The above results suggest that the inhibitory effect
of A23187 on TNF-stimulated PAI-1 synthesis occurs at a
posttranscriptional level. To further investigate this possibility, we
used protocol 2 for cell stimulation (Fig 1), in which A23187 treatment
was performed once the TNF-induced synthesis of PAI-1 mRNA was optimal,
ie, 7 hours (compare Fig 7A). Under these conditions, TNF alone (100
U/mL) increased the accumulation of PAI-1 in the conditioned EM by a
factor of 2.5 when compared with control cells (Table 2). In TNF-stimulated cells, delayed
treatment with A23187 (5 µmol/L) significantly reduced
PAI-1 accumulation almost to the level of control cells. This result
indicates that although both PAI-1 mRNA transcripts accumulated during
7 hours of TNF treatment, they were subsequently less efficiently
processed when A23187 was added.
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To examine how A23187 exerted its effect, just before A23187
stimulation we added a slight excess of EGTA to completely chelate the
extracellular calcium in the EM. As shown in Table 2, EGTA tended to
decrease the TNF-activated production of PAI-1 when
compared with standard calcium concentrations, although the difference
was not statistically significant. The inhibition by A23187 of the
PAI-1 production activated by TNF was not significantly
reversed by EGTA. This finding indicates that suppression of calcium
influx has no effect on the inhibition of TNF-induced PAI-1 synthesis
by A23187 and suggests that this compound exerted its
inhibitory effect through the depletion of internal calcium
stores. These results prompted us to study intracellular calcium
mobilization by fluorescence videomicroscopy analysis.
As shown in Fig 8, in cells stimulated
with TNF alone, A23187 induced a short and rapid increase in cytosolic
calcium concentration followed by a decrease and a plateau above the
basal level. When EGTA was added shortly before A23187, the magnitude
of the calcium peak was comparable, but the level of cytosolic calcium
rapidly returned to the basal level. When the addition of EGTA preceded
for a longer time (2 to 3 hours) the addition of A23187, the kinetics
of calcium mobilization exhibited a comparable profile to that when
EGTA was added shortly before A23187. Thus, A23187 mobilized calcium
still present in internal stores despite the prolonged absence of
extracellular calcium.
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At this stage of investigation, we propose that the A23187-induced depletion of internal calcium pools is responsible for the inhibition of PAI-1 synthesis that was initially activated by TNF. We therefore further investigated at which step of the posttranscriptional processing of PAI-1 mRNAs calcium acted.
In TNF-Stimulated Cells, A23187 Did Not Alter the Stability of
PAI-1 mRNAs
To identify the posttranscriptional step at which A23187 exerted
its effect, we investigated the stability of PAI-1 mRNA by using
protocol 2 (Fig 9). In TNF-stimulated
cells subsequently treated with A23187 and then with actinomycin D, the
level of each transcript did not change between 2 and 6 hours (bar 2
versus 6), indicating that A23187 treatment did not alter the stability
of the two PAI-1 mRNAs. In the presence of A23187, the level of PAI-1
mRNAs was higher than in cells stimulated with TNF alone. This was
observed 2 hours after treatment with A23187 (bar 2 versus 1, +53%)
and more marked after 6 hours (bar 4 versus 1, +90%). This result is
likely attributable to activation of transcription, as suggested by
experiments with actinomycin D, which showed that the levels of PAI-1
mRNAs analyzed at 6 hours were similar to those measured at 2
hours (bar 6 versus 2). Analysis of PAI-1 mRNAs at 6 hours
clearly showed that enhanced transcription preferentially affected the
2.3-kb form sufficiently to give a resulting pattern close to that
obtained when A23187 and TNF were added simultaneously
(compare with Fig 7). We then investigated whether this altered shift
between the two PAI-1 mRNA species could have any consequence on the
translational process.
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In TNF-Stimulated Cells, A23187 Altered the Polysomal Distribution
of PAI-1 mRNAs
The sedimentation behavior of mRNA on a sucrose gradient allows
the detection of alterations associated with translating ribosomes.
Cell lysates were fractionated on a linear 15% to 50% gradient of
sucrose and the PAI-1 mRNA sedimentation profile analyzed.
Protocol 2 of cell stimulation was used. Fig 10A shows that under TNF treatment,
most of the PAI-1 mRNAs sedimented with dense polysomes (75%
sedimented in fractions 11 to 14) that represent the actively
translating ribosomal pool. Both transcripts sedimented to the same
extent as active translating ribosomes. In untreated cells or cells
treated with A23187 alone, PAI-1 mRNAs showed sedimentation profiles
similar to those of TNF-stimulated cells, although less intense signals
were observed (data not shown). To prove that under our experimental
conditions of separation PAI-1 mRNAs were functionally bound to
actively translating ribosomes, to the cell lysate and the gradients we
added EDTA (final, 30 mmol/L) to fully chelate
MgCl2. This procedure usually disrupts polysomes into 80S
monosomes and releases the mRNA of interest, which sediments in the
lighter fractions mainly in the free form. Such treatment effectively
displaced and gathered PAI-1 mRNAs into the lightest fractions (100%
in fractions 1 to 6, with a maximum in fractions 3 and 4; data not
shown), attesting that the PAI-1 mRNAs that sedimented in the heaviest
fractions were engaged in the actively translating pool. When A23187
was added to TNF-stimulated cells, the absolute amount of PAI-1 mRNA
recovered was higher than that with TNF alone (Fig 10B), confirming the
above results that delayed addition of A23187 triggered PAI-1 gene
transcription. The alteration in transcript distribution was also
observed in the polysomal pool. When expressed as a percentage, it is
significant that a lower proportion of PAI-1 mRNA sedimented in the
translating pool (about 50% in fractions 11 to 14 versus 75% in
TNF-stimulated cells), whereas a higher proportion sedimented in less
dense fractions (50% in fractions 1 to 10 versus 25% in
TNF-stimulated cells), corresponding to various lighter forms,
including free and initiation forms. Interestingly, both 3.2- and
2.3-kb transcripts followed a parallel sedimentation profile, including
that of dense polysomes. The A23187-induced modifications of PAI-1 mRNA
sedimentation do not appear to be a general process, as judged by the
GAPDH mRNA sedimentation profile, which was not altered.
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Specificities of the Effects Observed
We used TG, which also empties internal calcium stores, though in
a different way than A23187.43 TG (0.5
µmol/L) added simultaneously with TNF (protocol 1)
decreased PAI-1 accumulation by a factor 3 when compared with TNF alone
(Fig 11). The change in the
distribution of the PAI-1 mRNA transcripts in TNF+TG–treated cells was
nearly the same as that observed in TNF+A23187–treated cells (compare
with Fig 7). To investigate whether the effect of A23187 was
specifically associated to TNF or more generally to proinflammatory
cytokines, we stimulated HUVECs with IL-1ß, which is also
known to stimulate in vitro PAI-1 synthesis in human
ECs.44 As shown in Fig 12, IL-1ß alone (10 U/mL) or added simultaneously with A23187
(protocol 1) induced effects on PAI-1 accumulation in the conditioned
EM that were comparable to those described above with TNF.
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Discussion |
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This work addresses the effect of cross-talk between intracellular calcium mobilization and TNF signaling on the synthesis of PAI-1 in human ECs. Whereas A23187 alone slightly enhanced both mRNA and protein PAI-1 levels, it abolished the TNF-activated PAI-1 accumulation in the EM. Several intracellular alterations, such as a sublethal state, an apoptotic process, proteolysis, or a defect in the secretory process induced by the combination of TNF and A23187, may account for the inhibitory effect. The results presented herein clearly allow us to rule out these possibilities. The viability of cells was further attested to by the fact that cells treated with TNF and A23187 reversibly recovered their ability to increase PAI-1 production in response to a second stimulation by TNF.
The suppression by A23187 of TNF-activated accumulation of PAI-1 in the conditioned EM is reflected by a lower level of intracellular PAI-1, which oriented our investigations at the intracellular level. A calcium-induced lower rate of gene transcription can be ruled out a priori, since the levels of PAI-1 mRNA are at least the same whether the cells are stimulated by TNF alone or by simultaneous addition of A23187 and TNF. Convincing support for an impaired posttranscriptional event affecting PAI-1 mRNAs comes from the results of the experiment in which A23187, added 7 hours after TNF, still induced its suppressing action despite the fact that the PAI-1 mRNA level was slightly enhanced. A23187-induced calcium mobilization has been shown to decrease TNF-induced inducible nitric oxide synthase synthesis by decreasing its mRNA stability.18 However, treatment with actinomycin D demonstrated that PAI-1 mRNA stability was not altered by any of the treatments. PAI-1 mRNAs have also been reported to be stable in TNF-treated HUVECs.45 A peculiar feature that could have accounted for the observed inhibition of PAI-1 synthesis was the significant shift between the 3.2- and 2.3-kb species. These two forms are produced by alternative polyadenylation sites,39 40 and changes in their distribution have already been described under different conditions of stimulation.46 47 The presence and length of the poly(A) "tail" are thought to modulate the mRNA translational rate (reviewed in Reference 4848 ). Therefore, we envisaged that the altered shift might have impaired in some way the translational efficiency of PAI-1 mRNAs, although both species are generally considered to be translated at the same efficiency, which has not been demonstrated so far. Herein we showed that whichever treatment was used, both 3.2- and 2.3-kb transcripts exhibited identical sedimentation profiles on sucrose gradients, especially in those fractions corresponding to the dense ribosomal pool that directs active translation. This finding supports the contention that both species are able to be translated at the same efficiency and indicates that the A23187-induced shift in the 3.2/2.3 ratio presumably has no functional consequences in terms of translational capacities of the two species.
A23187 induces both depletion and influx of calcium from internal stores. Chelation of extracellular calcium by EGTA did not prevent the A23187-induced inhibition of PAI-1 synthesis stimulated by TNF, indicating that calcium depletion itself of internal stores is sufficient to cause inhibition. In support of this, TG, which also empties endoplasmic calcium stores by inhibiting the repumping activity of Ca2+-ATPases, exerted comparable effects on TNF-stimulated PAI-1 synthesis. It is noteworthy that the A23187-induced inhibitory process we describe herein is specifically associated with the signaling event triggered by TNF. Indeed, cells treated with A23187 alone did not exhibit any alteration in the sedimentation profile of polysome-associated PAI-1 mRNAs, nor did they produce less amounts of PAI-1 protein when compared with control cells. This indicates that the release of calcium itself from endoplasmic stores does not affect the translation of PAI-1 mRNAs but negatively and specifically interacts with a process that is upregulated by TNF and eventually decelerates the TNF-activated PAI-1 mRNA translation. Regulation of translation usually occurs at the initiation stage, because this is the rate-limiting step (reviewed in References 49 and 5049 50 ) that also requires calcium sequestered in endoplasmic stores (reviewed in References 51 and 5251 52 ). In cells stimulated with TNF and A23187, the proportion of PAI-1 mRNA in less dense fractions at a given time was increased twofold when compared with TNF alone. This cannot be explained by a "tailing" phenomenon due to the higher absolute level of PAI-1 mRNA, because the sedimentation profiles of PAI-1 mRNAs of control, A23187-treated, and TNF-stimulated cells were identical despite a higher amount of PAI-1 mRNAs in the case with TNF. This event is likely a consequence of a reduced number of ribosomes recruited on the mRNA that is indicative of a slower initiation process.49 53 Interestingly, some initiation factors are regulated either after phosphorylation by various stimuli, including TNF54 55 56 or by the calcium content of endoplasmic stores.57 It is, however, premature to assert that alterations in the initiation step can fully account for the inhibition of PAI-1 protein synthesis. Although elongation is usually not considered the rate-limiting step in translation, there are some indications that in HUVECs, calcium mobilization can regulate this process through the activity of calcium/calmodulin–dependent protein kinase III.58 59 Thus, we propose a tentative model in which TNF not only activates PAI-1 gene transcription but also PAI-1 mRNA translation to coordinate transcriptional and translational stages. The initiation and/or elongation steps would be regulated by a TNF-dependent phosphorylation state of calcium-dependent factor(s) and therefore would be inhibited if the calcium content sequestered in endoplasmic pools were lowered. The fact that EGTA only slightly decreased PAI-1 synthesis in cells stimulated by TNF alone deserves some comment. According to our tentative model, this implies that despite the presence of extracellular EGTA, calcium was still present in endoplasmic pools, thus allowing the translation of TNF-induced PAI-1 mRNA to proceed at an almost-normal rate. Analysis of calcium mobilization by videomicroscopy indeed revealed that even after 2 to 3 hours in the presence of extracellular EGTA, calcium was still sequestered in internal stores, indicating that these stores efficiently resisted extracellular EGTA.
Intracellular signaling triggered by calcium mobilization is very complex and can activate several pathways. Beside its posttranscriptional effect, calcium mobilization moderately increased PAI-1 mRNA levels. This phenomenon was already observed in U937 cells but with much more pronounced effects.16 Because an enhanced stabilizing effect does not account for our observations, one likely explanation is that the increase in cytosolic calcium triggers transcription via calcium/calmodulin–dependent protein kinases,25 26 which when cells are stimulated by TNF, may cross-talk with a TNF-dependent kinase pathway activating PAI-1 gene transcription.42
It is likely that the mode of inhibition of A23187 is not specific for PAI-1; on the other hand, it is obvious that it did affect a small pool of proteins. First, [3H]leucine incorporation in the entire protein pool only tended to decrease, and second, the sedimentation profile of polysomes analyzed at 254 nm did not reveal any significant modifications regardless of which treatments were applied, a situation that is not the case when global translation is drastically altered. The downregulating effect we described for TNF appears also typical of proinflammatory cytokines, because IL-1ß–induced PAI-1 production was also decreased by A23187 to a comparable extent. Taken together, present and previous data16 17 18 19 point out the importance of integrating cytokine signaling with other signaling pathways, at present intracellular calcium mobilization, when studying proteins whose synthesis is activated by cytokines.
In conclusion, our results demonstrate that calcium-mobilizing agents in HUVECs suppress the increase in PAI-1 synthesis triggered by proinflammatory cytokines. This effect appears closely linked to the calcium emptying of endoplasmic stores, which results in impairment of the translation of the two transcripts, at least at the initiation step. This process in ECs may provide a regulatory mechanism of PAI-1 synthesis, inasmuch as these cells are often subjected, under pathophysiological situations (such as atherosclerosis), to several stimuli, including cytokines and calcium homeostasis disruption.
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Acknowledgments |
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This work was supported by funds from INSERM, DRED, and Fondation de France. F. Peiretti is a recipient of the Ministère de la Recherche et de l'Enseignement Supérieur. The authors wish to acknowledge the skilful technical assistance of M. Delézay, B. Bonardo, and A. Olivi (cell culture); O. Geel (ELISA); and D. Nivière for secretarial help. Drs R. Lijnen and P. Declerck (Leuven, Belgium) kindly provided the PAI-1 monoclonal antibodies. We are indebted to A.M. Benoliel (INSERM U387, Marseille) for videomicroscopy analyses, the Laboratoire de Biochimie (CHU-Timone, Marseille) for lactate dehydrogenase assays, and INSERM U38 (Marseille) for the densitometric scanning. We are grateful to P. Declerck, J. Iovanna (INSERM U315, Marseille), and F. Berenbaum (Hôpital St Antoine, Paris) for their helpful advice and suggestions.
Received May 28, 1996; accepted October 21, 1996.
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