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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1550-1560

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1550-1560.)
© 1997 American Heart Association, Inc.

Articles

Intracellular Calcium Mobilization Suppresses the TNF-{alpha}–Stimulated Synthesis of PAI-1 in Human Endothelial Cells

Indications That Calcium Acts at a Translational Level

Franck Peiretti; Marie-Christine Alessi; Mireille Henry; Francine Anfosso; Irène Juhan-Vague; ; Gilles Nalbone

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.


*    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-{alpha} (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


*    Introduction
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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-{alpha}–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.


*    Methods
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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-{alpha} (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 1Down). 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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  		Figure 1. Schematic representation of the two protocols of HUVEC stimulation. Protocol 1 refers to simultaneous treatment with TNF and A23187 and protocol 2 to the delayed treatment with A23187 of TNF-stimulated cells. In some experiments performed with protocol 1, TNF was replaced by IL-1ß and A23187 by TG. In some experiments performed with protocol 2, actinomycin D was added in a single dose 20 minutes before t=2 hours. The different doses of cytokines and calcium-mobilizing agents and time of analysis are indicated in the corresponding figures.

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 {approx}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.


*    Results
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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 2Down). Therefore, in all subsequent studies, the concentration of TNF was fixed at 100 U/mL. As shown in Fig 2Down, 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 3Down). 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 3Down). 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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  		Figure 2. Time-dependent accumulation of PAI-1 secreted into the conditioned EM from TNF-stimulated cells. TNF was added in a single dose of 100 U/mL and conditioned EM collected at the indicated times. Values are mean±SD from three separate experiments, each performed in triplicate (n=9). The differences in means between the TNF vs control (unstimulated) cells were significant from 14 to 20 hours (P<.01). Inset, Dose-dependent effect of TNF on PAI-1 accumulation measured at 20 hours.



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  		Figure 3. Effect of simultaneous treatment with TNF and A23187 on accumulation of PAI-1 secreted into the conditioned EM. HUVECs were stimulated with TNF alone at 100 U/mL (hatched bar) or by simultaneous addition of TNF and increasing concentrations of A23187 (1-10 µmol/L, shaded bars) according to protocol 1. After 30 minutes, the EM of cells treated or not with A23187 was aspirated, and the cells were washed twice with fresh EM. TNF was then reintroduced in the same single dose. Conditioned EM were collected at 20 hours. Control cells (C, black bar). Values are mean±SD from three separate experiments, each performed in triplicate (n=9). **Significant at P<.01 vs TNF. Inset, Time-dependent accumulation of PAI-1 in conditioned EM from TNF- and TNF+A23187–stimulated cells. TNF was at 100 U/mL and A23187 at 5 µmol/L.

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 4Down, 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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  		Figure 4. Effect of simultaneous treatment with TNF and A23187 on intracellular levels of PAI-1. HUVECs were stimulated with TNF (100 U/mL) or TNF and A23187 (5 µmol/L) as described in Fig 3Up. At the indicated times, cells were washed, trypsinized, and then lysed in PBS–Triton X-100 (0.1%) buffer for 15 minutes at 4°C. The lysates were centrifuged and the supernatants saved for PAI-1 determination. Values are mean±SD from three separate experiments, each performed in duplicate (n=6). **Significant at P<.01 vs TNF-stimulated cells.

In TNF-Stimulated Cells, A23187 Did Not Induce Proteolysis or Apoptosis
As shown in Table 1Down, 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 1Down). 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 5Down) 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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  		Table 1. Effect of Simultaneous Treatment With TNF and A23187 on Lactate Dehydrogenase Release and Global Protein Turnover



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  		Figure 5. SDS–polyacrylamide gel electrophoresis of immunoprecipitates of [35S]methionine-labeled PAI-1 obtained from HUVECs subjected to pulse-chase conditions. The effect of TNF and TNF+A23187 treatments on intracellular degradation of PAI-1 in HUVECs was investigated by pulse-chase experiments using protocol 2. Experimental conditions of pulse-chase and immunoprecipitation are detailed in "Methods." PAI-1 immunoprecipitates were analyzed by SDS–polyacrylamide gel electrophoresis at the time of A23187 addition (t=0) and then 0.5 and 1 hour later. Lanes 1, 2, 4, TNF alone (100 U/mL) at t=0, t=0.5, and t=1 hour, respectively; lanes 3 and 5, TNF+A23187 (5 µmol/L) at t=0.5 and t=1 hour, respectively. The PAI-1 position was verified by purified PAI-1 and a protein-standard calibration kit.

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 6Down, 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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  		Figure 6. Agarose gel electrophoresis of DNA from HUVECs treated with TNF and A23187. Stimulation was done according to protocol 1 as indicated in Fig 3Up. At 18 hours, HUVECs were lysed and DNA extracted as described in "Methods." Lane M, DNA size markers from 1500 to 100 bp; lane A, positive control of apoptosis obtained by incubating cells with a mixture of actinomycin D (5 µg/mL), TNF (1000 U/mL), and A23187 (5 µmol/L) for 6 hours; lane 1, unstimulated control cells; lane 2, cells stimulated with TNF; and lane 3, cells treated simultaneously with TNF+A23187. TNF was at 100 U/mL and A23187 at 5 µmol/L.

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 1Up). 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 7ADown). A23187 alone (5 µmol/L) exerted a slight transient increase in PAI-1 mRNA at 6 to 7 hours (Fig 7ADown). 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 7ADown). 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 7ADown). 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 7BDown). 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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  		Figure 7. Time-dependent effect of simultaneous treatment with TNF and A23187 on PAI-1 mRNA transcript levels. Stimulation was performed according to protocol 1 as in Fig 3Up. A, Densitometric measurements of total PAI-1 mRNA (3.2 kb and 2.3 kb) levels in control, A23187-, TNF-, and TNF+A23187–stimulated cells. TNF was at 100 U/mL and A23187 at 5 µmol/L. Values are mean±SD from two experiments, each performed in duplicate (n=4). B, Individual levels of each PAI-1 mRNA transcript in TNF- and TNF+A23187–stimulated cells. Upper, Northern blot; lower, densitometric analysis of a Northern blot representative of two separate experiments. All densitometric measurements were normalized to GAPDH mRNA.

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 1Up), in which A23187 treatment was performed once the TNF-induced synthesis of PAI-1 mRNA was optimal, ie, 7 hours (compare Fig 7AUp). Under these conditions, TNF alone (100 U/mL) increased the accumulation of PAI-1 in the conditioned EM by a factor of {approx}2.5 when compared with control cells (Table 2Down). 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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  		Table 2. Effects in TNF-Stimulated Cells of Delayed Addition of A23187 on PAI-1 Accumulation in Conditioned EM

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 2Up, 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 8Down, 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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  		Figure 8. Videomicroscopy analysis of intracellular calcium mobilization in HUVECs induced by A23187. HUVECs were stimulated with TNF (100 U/mL) for 6 hours, incubated with fluo 3-AM (1 µmol/L) for 25 minutes, and then stimulated with A23187 (5 µmol/L) as indicated by the arrow. Line 1, EM with standard calcium (1.3 mmol/L); line 2, calcium in EM chelated with EGTA 2-3 minutes before A23187 stimulation; and line 3, calcium in the EM chelated 2-3 hours before A23187 stimulation. Values are expressed as n-fold increases above the level of fluorescence in each cell measured a few seconds before addition of A23187. Each point represents the mean of the recording of 5-8 individual cells with an SD that did not exceed 10% of the mean. This figure is representative of two separate experiments.

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 9Down). 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 7Up). 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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  		Figure 9. Effect of delayed addition of A23187 and actinomycin D in TNF-stimulated cells on PAI-1 mRNA transcript levels. Protocol 2 was applied. Columns 1, 3, 5, cells stimulated with TNF alone (100 U/mL); columns 2, 4, 6, cells stimulated with TNF and then with A23187 (5 µmol/L). The time of A23187 introduction was considered as t=0. Conditioned EM of cells treated or not with A23187 was eliminated at t=30 minutes; cells were washed twice with fresh EM and then incubated in fresh EM alone. In the experiments with actinomycin D, the antibiotic (5 µg/mL) was added 20 minutes before t=2 hours (bars 5 and 6); 2 and 6 hours indicate the times at which RNA was extracted. Upper, Northern blot; lower, corresponding densitometric measurement of the Northern blot normalized to GAPDH mRNA. This figure is representative of three separate experiments.

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 10ADown 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 10BDown), 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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  		Figure 10. Sucrose gradient sedimentation profile of ribosome-associated PAI-1 mRNA isolated from HUVEC lysates. HUVECs were stimulated with TNF (100 U/mL) for 7 hours (A) and then treated with A23187 (5 µmol/L) for 30 minutes (B) according to protocol 2. HUVECs were trypsinized and lysed 4 hours after A23187 treatment, and the supernatant of the centrifuged lysate was submitted to sucrose gradient (15-50%) sedimentation. Sedimentation proceeded from left to right. The absorbance at 254 nm was recorded during collection (full line). Fractions of {approx}0.8 mL were collected and submitted to Northern blot analysis (insets) for PAI-1 and GAPDH mRNA identification. Examination under UV of 18S and 28S spots from total RNA of cell lysates attested that comparable amounts of RNA had been subjected to ultracentrifugation. The joined histograms represent the densitometric scanning of PAI-1 mRNAs. Each histogram is expressed as the % of total PAI-1 mRNA recovered in the gradient. The figure is representative of two separate experiments.

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 11Down). 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 7Up). 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 12Down, 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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  		Figure 11. Effect of simultaneous treatment with TNF and TG on PAI-1 synthesis. Protocol 1 of stimulation was applied, except that A23187 was replaced by TG. PAI-1 antigen accumulation in the conditioned EM was allowed to proceed for 18 hours. From left to right are control cells, cells stimulated with TNF alone, and cells treated simultaneously with TNF and TG. TNF was at 100 U/mL and TG at 0.5 µmol/L. Values are mean±SD from three separate experiments, each performed in duplicate (n=6). **Significant at P<.01 vs TNF. Inset, 1, 2, 3 in the same order as the bars represent the level of PAI-1 mRNA transcripts analyzed at 7 hours.



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  		Figure 12. Effect of simultaneous treatment with IL-1ß and A23187 on PAI-1 antigen accumulation in conditioned EM. Protocol 1 of stimulation was applied, except that TNF was replaced by IL-1ß. PAI-1 antigen accumulation in conditioned EM was allowed to proceed for 18 hours. From left to right are control cells, cells stimulated with IL-1ß alone (10 U/mL), and cells treated simultaneously with IL-1ß and A23187 (5 µmol/L). Values are mean±SD from three separate experiments, each performed in duplicate (n=6). **Significant at P<.01 vs IL-1ß.


*    Discussion
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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.


*    Selected Abbreviations and Acronyms
 
EC = endothelial cell
ELISA = enzyme-linked immunosorbent assay
EM = experimental medium
FCS = fetal calf serum
fluo 3-AM = fluo 3 acetoxymethyl ester
HUVEC = human umbilical vein endothelial cell
IL = interleukin
MEM = minimal essential medium
PAI-1 = type 1 plasminogen activator inhibitor
TG = thapsigargin
TNF = tumor necrosis factor
TPA = tissue plasminogen activator


*    Acknowledgments
 
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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