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J Biol Chem, Vol. 275, Issue 5, 3081-3087, February 4, 2000
Tumor Necrosis Factor Up-regulates in an Autocrine Manner the
Synthesis of Plasminogen Activator Inhibitor Type-1 during Induction of
Monocytic Differentiation of Human HL-60 Leukemia Cells*
Sophie
Lopez ,
Franck
Peiretti,
Bernadette
Bonardo,
Irène
Juhan-Vague, and
Gilles
Nalbone§
From the INSERM EPI 99-36, Laboratoire d'Hématologie,
Faculté de Médecine, 27 Bd. Jean Moulin,
13385 Marseille Cedex 5, France
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ABSTRACT |
Tumor necrosis factor- (TNF ) critically
regulates several cellular functions during monocyte/macrophage
differentiation. We therefore investigated during the phorbol ester
(phorbol 12-myristate 13-acetate (PMA))-induced monocyte/macrophage
differentiation of the human HL-60 leukemia cells, if TNF
contributed to plasminogen activator inhibitor type-1 (PAI-1) synthesis
that is initiated by a protein kinase C -extracellular
signal-regulated kinase 2-dependent pathway (Lopez, S.,
Peiretti, F., Morange, P., Laouar, A., Fossat, C., Bonardo, B.,
Huberman, E., Juhan-Vague, I., and Nalbone, G. (1999) Thromb.
Haemostasis 81, 415-422). Following PMA treatment, the level of
TNF mRNA strongly increased and appeared earlier than PAI-1
mRNA. An anti-TNF antibody significantly inhibited the
PMA-induced PAI-1 mRNA and protein levels. The recombinant human
TNF , which is inactive on native HL-60 cells in terms of PAI-1
synthesis, optimally potentiates it once HL-60 cells are committed into
the differentiation process. The use of 1) the HL-525 cell line, a
clone issued from HL-60 cells rendered resistant to PMA-induced
differentiation, and 2) the transforming growth factor -1/vitamin D3
differentiative mixture confirmed the relationships between the
induction of differentiation and the potency of TNF to up-regulate
PAI-1 synthesis. In conclusion, we showed that during the induction of
monocyte/macrophage differentiation, TNF and PAI-1 gene expressions
are activated and that synthesized TNF up-regulates and prolongs, in
an autocrine manner, the synthesis of PAI-1.
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INTRODUCTION |
The expression of
PAI-1,1 a major inhibitor of
fibrinolysis, is enhanced in atherosclerotic plaque (1-5) and
co-localizes, in part, with macrophages (2, 4, 6) that are central cell
types in atherosclerosis. These observations are consistent with the
fact that either macrophages isolated from atherosclerotic plaque (7)
or monocyte-transformed foam cells (8) significantly release PAI-1.
This emphasizes the essential role of resident macrophages in
regulating cellular events related to the pericellular proteolytic
activity in the vessel wall. The risk of intravascular thrombotic
events, associated with an excess of circulating (9) or arterial (10)
PAI-1, is well established in the insulin resistance syndrome. However,
the impact of PAI-1 on vascular remodeling and plaque stability appears
quite complex to delineate. By regulating plasmin-mediated matrix
metalloproteinase activity, PAI-1 may alter the structure of the
extracellular matrix and associated cellular events. These include, for
example, regulation of extracellular matrix degradation in a model of
aneurysm in rat (11) and smooth muscle cell migration in a model of
vascular injury in PAI-1 gene knock-out mice (12). In addition, a
non-anti-proteolytic function of PAI-1 was proposed from in
vitro data. An excess of PAI-1 was shown to prevent attachment of
different types of cells, including monocytes, by competing for the
vitronectin binding site with uPAR and v 3
(13-16). Contrarily, endogenously secreted PAI-1 was shown to
stabilize adhesion of the fibrosarcoma cell line HT-1080 (17, 18).
Which of the anti- or pro-adhesive functions of PAI-1 can be considered
as adverse during atherosclerosis is still an open question.
The human leukemia cell line HL-60 is a powerful and convenient model
to elucidate the regulation and function of factors that coordinate the
monocyte/macrophage adherent phenotype and pericellular proteolytic
activity. When treated with differentiative agents such as phorbol
esters, these cells develop the adherent macrophage-like phenotype (19)
and synthesize PAI-1 (20-22). We recently identified the early
signaling pathway activating PAI-1 synthesis during the induction of
differentiation in these cells, which is characterized by a linear
cascade involving PKC -MAPKK-MAPKp42 (22). In HepG2 cells treated
with phorbol 12-myristate 13-acetate (PMA), this pathway leads to the
c-Jun homodimer binding at the 58 to 50 region of the PAI-1
promoter (23). TNF , which is released by activated macrophages, is
an important mediator in the differentiation process of leukocytes
(24). This is also a major inducer of PAI-1 synthesis in various
differentiated cells (25, 26). The synthesis of PAI-1 in human immature
progenitor of monocytes and in isolated monocytes is very low (20, 22, 27, 28) and rarely activated by TNF (22, 29). Macrophages isolated
from atheromatous plaque, however, produce more PAI-1 than monocytes
isolated from blood of the same donor (7) strongly suggesting a
differentiated state-related synthesis of PAI-1 in this type of cell.
HL-60 cells differentiated by PMA also synthesize TNF (30), which
was recently demonstrated to up-regulate, in an autocrine manner, the
synthesis of 92-kDa gelatinase via 5 1 integrin expression (31). Because a close relationship exists between
PAI-1 and pericellular proteolytic activity, this drove us to use this
model to examine if TNF contributes to PAI-1 synthesis during
induction of leukocyte differentiation and if so, how it exerts this regulation.
Results showed that, during the early steps of the monocytic
differentiation program, TNF and PAI-1 mRNA levels increased. The synthesized TNF up-regulates and prolongs, in an autocrine manner, elevated PAI-1 mRNA levels resulting in enhanced protein antigen synthesis and secretion.
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EXPERIMENTAL PROCEDURES |
Materials--
Phorbol esters (PMA), phorbol dibutyrate (PdBu),
and PD 098059 were purchased from Alexis (Coger). PKC inhibitor Ro
31-8220 was provided by Dr. Bradschaw (Roche Laboratories). Monoclonal antibodies (12A4 and 15H12) specific for human PAI-1 were generously given by Dr. P. Declerck (Center for Thrombosis and Vascular Research, Leuven, Belgium). Monoclonal antibody against TNF were from R&D Systems. Recombinant human TGF- 1 (rhTGF- 1) and TNF (rhTNF ) were from Amersham Pharmacia Biotech. The Moloney-murine leukemia virus
reverse transcriptase and its appropriate buffer were purchased from
Life Technologies, Inc. Taq polymerase and its appropriate buffer were from Bioprobe. Other molecular biological products (dNTP,
random hexaprimers, RNasin® , and appropriate buffers)
were from Promega. Phorbol esters, PKC, and MAPKK inhibitors were
stored at 20 °C in Me2SO. All trans
retinoic acid (RA) and 1 ,25-dihydroxyvitamin D3 (D3) were stored at
20 °C in ethanol. Appropriate dilutions were made in warm culture
medium in such a way that the final Me2SO or ethanol concentration in the presence of cells did not exceed 0.1% (v/v). Proper controls made with similar Me2SO or ethanol
concentrations were without effects on the parameters studied.
Cell Culture--
The human promyelocytic HL-60 leukemia cell
line and the HL-525 cell line that is resistant to PMA-induced
differentiation (32) were kindly provided by Pr. E. Huberman (Argonne
National Laboratory, Argonne, IL). HL-60 and HL-525 cells were grown in RPMI containing 10% fetal calf serum as already described (22). Cells
in 72-h-old culture medium were resuspended for 1 h in fresh medium and then treated with differentiative agents.
RNA Extraction and Semi-quantitative RT-PCR Analysis--
Total
RNA extraction (extraction kit for total RNA, RNeasy from Quiagen) and
cDNA synthesis were performed as recently described (22). The
amplified fragment for human PAI-1 (GenBankTM accession
number X04744) is 284 bp, base position 10977-11260 and that for human
TNF (GenBankTM accession number M10988) is 296 bp, base
position 761-1056. The amplified fragment of TNF-RI (p55)
(GenBankTM accession number X55313) is 261 bp, base
position 786-1046 and that of TNF-RII (GenBankTM accession
number M55994) is 391 bp, base position 594-984. In PMA-treated cells,
eukaryotic elongation factor (eEF1 ) was observed to be a stable
house keeping gene, whereas in TGF- 1/D3-treated HL-60 cells,
-actin was preferred. The amplified fragment for eEF1
(GenBankTM accession number X03558) is 289 bp, base
position 382-670 and is 388 bp for -actin (GenBankTM
accession number X00351), base position 379-766. PCR was performed on
a Perkin-Elmer thermocycler (GeneAmp 2400). We selected a number of 25 cycles for TNF and its receptors, PAI-1, and -actin cDNAs and
a number of 18 cycles for eEF1 . Specificity of the amplified fragment was assessed by the demonstration that appropriate restriction enzymes generated the expected cleavage fragments. PCR started for 2 min at 95 °C followed by cycles consisting of: 60 s at 58 °C
(for PAI-1, eEF1 , and -actin) or 57 °C (for TNF and its receptors), 90 s at 72 °C, and 45 s at 97 °C.
Amplification was terminated after 5 min at 72 °C. Products were
visualized and photographed under UV radiation following gel-agarose
(2%) electrophoresis.
Protein Assays--
Total proteins of cell lysates were assayed
according to specifications of the bicinchoninic acid protein assay kit
from Sigma. PAI-1 antigen assay was performed on supernatants from
conditioned culture medium and on cell lysates (Triton X-100 0.1%
final) by enzyme-linked immunosorbent assay as described by Declerck
et al. (33). TNF antigen assay was performed on culture
supernatants according to the specifications provided with the
enzyme-linked immunosorbent assay kit (Coulter-Immunotech).
Flow Cytometry Analysis--
Expression of TNF receptors p55
(RI) and p75 (RII) were analyzed by flow cytometry. The experimental
protocol was comparable to that previously described for the urokinase
receptor (27). Briefly, HL-60 cells (105) were first
acid-treated (glycine buffer, pH 3.0, 1 min at 37 °C) to remove
bound TNF . Cells were then incubated with fluorescein isothiocyanate-conjugated monoclonal antibody against TNF RI or RII
(R&D Systems) and washed. Fluorescein isothiocyanate-labeled cells were
analyzed on a XL-cytofluorograph (Coulter Electronics Inc.) at 488 and
525 nm, corresponding to excitation and detection wavelengths, respectively.
Statistics--
Each experiment was performed in duplicate or
triplicate. Results are expressed as mean ± S.D. Comparisons were
analyzed by an analysis of variance (ANOVA) test, and significance was
calculated at p < 0.05 or p < 0.01 using the Scheffe F-test.
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RESULTS |
PMA Increases TNF mRNA Level Earlier than That of
PAI-1
In nonstimulated HL-60 cells, the level of TNF mRNA was low
(Fig. 1A). When HL-60 cells
were treated with 20 nM PMA, the TNF mRNA level rose
rapidly. The optimum expression was attained 2 h after PMA
addition. The increase in TNF mRNA level was transient since
24 h after PMA addition, the level was below that of nonstimulated cells (Fig. 1A). TNF antigen accumulation increased by a
factor 3.6 in the culture medium of HL-60 cells treated with PMA (Fig. 1B). The PAI-1 mRNA level (Fig. 1A) was not
detectable in basal conditions but was strongly induced 4 h after
PMA addition and remained elevated over 24 h. PAI-1 antigen
accumulation was dramatically enhanced at 24 h compared with
nontreated HL-60 cells (Fig. 1B).

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Fig. 1.
Time-dependent synthesis of PAI-1
and TNF in PMA-treated HL-60 cells. HL-60
cells (1 × 106/ml) were treated with PMA 20 nM. A, analysis by RT-PCR of the mRNA levels
at the indicated times. The levels of PAI-1 and TNF mRNAs in
untreated cells did not show any significant variations throughout the
experiment when compared with those shown at t = 0. This figure is representative of two separate experiments;
B, PAI-1 and TNF antigen accumulation in the culture
medium measured at 24 h in untreated (hatched bars) and
PMA-treated (solid bars) cells. Values are mean ± S.D.
(n = 6, three separate experiments performed in
duplicate); PMA values are significantly different versus
control at p < 0.01.
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Endogenous TNF Up-regulates PMA-induced PAI-1 Synthesis
In view of the above results and because PAI-1 is a
TNF -responsive gene in various differentiated cells, we then
examined if the synthesis of PAI-1 was attributable only to the
activation of the PKC -MAPKK-MAPKp42 pathway (22) or if the released
TNF contributed to the synthesis of PAI-1. HL-60 cells were
simultaneously incubated with a neutralizing antibody against human
TNF and PMA. Preliminary experiments revealed that the optimal
effect was obtained at 2.5 µg/ml anti-TNF . We used 10 µg/ml to
ensure complete quenching of released TNF . As shown in Fig.
2A, anti-TNF significantly
lowered the level of PAI-1 mRNA 4 h after PMA addition indicating that endogenously released TNF is already active at this
time. This inhibition strongly persisted at 10 h. The
intracellular level of PAI-1, measured 8 h after PMA addition, was
reduced by 41% (from 6.9 ± 2.1 down to 4.06 ± 2.0 ng/mg
proteins), whereas PAI-1 antigen accumulation in the culture decreased
by 65% at 24 h (Fig. 2B). IgG1 (10 µg/ml) used as a
control isotype in anti-TNF studies did not inhibit PMA-induced
PAI-1 synthesis and even tended to increase it by 20% (not shown).
This increase could be explained by the fact that in HL-60 cells, IgG
immunoglobulins activate the production of TNF (34). The IgG-induced
TNF release might in turn potentiate PAI-1 synthesis once
differentiation is initiated by PMA. Morphological examination of HL-60
showed that the addition of anti-TNF tended to reduce the spreading
of cells, which appear more rounded when compared with PMA-treated
cells (Fig. 3, A-C). Twenty
four hours after PMA addition, the percentage of adherent cells was
95 ± 3% and 62 ± 10% when anti-TNF was added. These results indicate that, in PMA-stimulated HL-60 cells, endogenous TNF
up-regulates, in an autocrine manner, the PAI-1 synthesis.

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Fig. 2.
Time-dependent effects of
anti-TNF and rhTNF on
PAI-1 synthesis in PMA-treated HL-60 cells. HL-60 cells (1 × 106/ml) were treated with PMA alone or simultaneously with
anti-TNF (10 µg/ml) or rhTNF (20 units/ml). A,
analysis by RT-PCR of the PAI-1 mRNA levels at the indicated times.
This figure is representative of two separate experiments;
B, PAI-1 antigen accumulation in the culture medium measured
at 24 h. Values are mean ± S.D. (n = 6, two
separate experiments performed in triplicate); PMA values are
significantly different at p < 0.05 versus
PMA + TNF and p < 0.01 versus PMA + anti-TNF .
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Fig. 3.
Microscopic examination of HL-60 and HL-525
cells 24 h after various treatments. Upper, HL-60
cells; A, nontreated; B, PMA; C, PMA + anti-TNF ; D, PMA + rhTNF . Lower, HL-525
cells; E, nontreated; F, PMA + rhTNF ;
G, RA + PMA; H, RA + PMA + rhTNF . Morphology
of HL-525 cells treated by RA, rhTNF , or RA + rhTNF (not shown)
are similar to that of nontreated cells. Magnification × 40.
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The Up-regulating Effect of TNF on PAI-1 Synthesis Is Linked to
the Induction of Differentiation
rhTNF Activates PAI-1 Synthesis in PMA-treated HL-60
Cells--
rhTNF did not induce any changes in the accumulation of
PAI-1 antigen from native HL-60 cells, which did not exceed 1 ng/106 cells (Fig. 2B) at 24 h. When
rhTNF was simultaneously added with PMA, it induced a modest
increase in PAI-1 antigen level. This was characterized by an increase
in the intracellular compartment of 18% after 8 h (from 6.9 ± 2.1 to 8.3 ± 2.4 ng/mg proteins) and 55% in the culture
medium after 24 h (Fig. 2B). To rule out possible
endotoxin contamination, rhTNF was boiled for 5 min and added on
PMA-treated cells. No enhancement of PAI-1 synthesis could be observed
in these conditions. The addition of rhTNF tended to increase the
spreading of adherent cells (Fig. 3D) but did not change the
PMA-induced percentage of adherent cells. The increase in PAI-1
synthesis was not detected at the mRNA level (Fig. 2A).
The moderate effect of rhTNF on PAI-1 synthesis could be due to the
rapid down-regulation of the expression of TNF receptors I and II
following PMA treatment as described previously in U937 cells (35) and
HL-60 cells (36). To investigate this possibility, we examined TNF
receptor (RI and RII) expression by RT-PCR and flow cytometry. As shown
in Fig. 4A, the level of RI
mRNA progressively increased the first 14-20 h then returned to
the basal level at 24 h. The RII mRNA basal level, higher than
RI, slightly decreased at 6 h and then was strongly enhanced until
20 h. It then decreased at 24 h. Surface expression of RI and
RII both increased with time peaking at 9 h (Fig. 4B).
The transient increase observed in untreated cells is likely the result
of the renewal of the 72-h-old culture medium, because the basal
expression of RI and RII was recovered around 36 h after culture
medium renewal (not shown). However, this transient increase was less
pronounced in PMA-treated than in untreated HL-60 cells. This is in
line with previous data showing that PMA rapidly down-regulates RI and
RII surface expression (35, 36), but is not strong enough in our experimental conditions to induce an absolute decrease when compared with their levels at t = 0.

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Fig. 4.
Time-dependent surface expression
of TNF-RI and TNF-RII in PMA-treated HL-60 cells. HL-60 cells were
treated with PMA as in Fig. 1. A, analysis by RT-PCR of the
TNF RI and TNF RII mRNA levels at the indicated times. This figure
is representative of two separate experiments B, flow
cytometry determination of surface expression of TNF-RI
(squares) and TNF-RII (circles) in PMA-treated
(closed symbols) or not (open symbols) cells at
indicated times. Values are mean ± S.D. (n = 4, two separate experiments each performed in duplicate).
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The lack of effect of rhTNF on PAI-1 synthesis observed on native
undifferentiated HL-60 cells is not linked to some defect in TNF
receptors, because they are expressed in a constitutive manner as we
demonstrated above and are operational in these cells (37, 38). This
led us to suggest the existence of a functional link between the
differentiation state and the potency of TNF to activate PAI-1 gene
expression. We thus evaluated the relationship between the
time-dependent monocyte/macrophage differentiation and the
potency of TNF to activate PAI-1 synthesis. To specifically evaluate
the effect of rhTNF from the global PMA effect, we treated HL-60
cells with a metabolizable phorbol ester (phorbol dibutyrate) for
various times (i.e. 3, 6, 14, 24, 36, and 48 h after
initial PdBu addition) at which conditioned medium was discarded. The cells were washed and then either stimulated or not (control) by
rhTNF for 24 h. As shown in Fig.
5, the enhancing effect of rhTNF
appears optimal 14-24 h after the induction of monocytic differentiation and then declines progressively but remains higher than
that of native undifferentiated HL-60 cells stimulated by rhTNF
alone. It should be mentioned that these results cannot be compared
with those of the PMA-induced up-regulating effect described in Fig. 2.
Indeed, once the PdBu-containing medium is removed, intracellular PdBu
is rapidly degraded, which stops cellular differentiation related
events. These results show that rhTNF activates PAI-1 synthesis once
HL-60 cells have been "primed" by a differentiative stimulus.

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Fig. 5.
Effect of rhTNF on
PAI-1 synthesis in HL-60 cells treated for various times by PdBu.
HL-60 cells were treated with PdBu (40 nM). At the
indicated times (3-48 h), conditioned medium was discarded, and cells
were rinsed with warm culture medium and resuspended without
reintroducing PdBu. rhTNF (20 units/ml) was added (hatched
bar) or not (gray bar). PAI-1 was measured in the
culture medium 24 h after rhTNF addition. Values are mean ± S.D. (n = 4, two separate experiments performed in
duplicate).
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Relationships between TNF and PAI-1 Synthesis in the HL-525 Cell
Line Resistant to PMA-induced Differentiation--
The HL-525 cell
line was cloned from HL-60 cells rendered resistant to PMA-induced
monocytic differentiation after long term PMA treatment. This results
in a down-regulation of PKC expression (32, 39). Restoration of PMA
responsiveness in terms of monocyte/macrophage differentiation can be
obtained by pretreatment of HL-525 cells with RA that re-induces PKC
gene expression (40).
Fig. 6A shows that, in HL-525
cells, the level of TNF mRNA was slightly enhanced by PMA alone,
although it was lower and appeared later than in HL-60 cells. Treatment
of HL-525 cells with RA alone had no effect on TNF mRNA levels,
except a slight transient increase at 6-8 h. Clearly, RA pretreatment
allows PMA to progressively and strongly enhance TNF mRNA
levels. It should be noticed that the optimum level of TNF mRNA
significantly increased much later than in HL-60 cells (10 versus 2 h). The amount of TNF antigen secreted into
the culture medium (Fig. 6B) showed that, in comparison with
control HL-525 cells, PMA modestly enhanced TNF antigen
accumulation, whereas RA alone had no marked effect. The combination of
RA + PMA increased the antigen level of TNF in the culture medium.
This level was 60% higher than that from HL-60 cells (cf.
Fig. 1B), and the increase continued for 48 h.

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Fig. 6.
Time-dependent analysis of
TNF synthesis in HL-525 cells. HL-525
cells (0.2 × 106/ml) were treated or not for 72 h by RA (1 µM). At t = 0 they were
suspended in fresh medium at 1 × 106/ml with or
without RA (0.5 µM) and were stimulated or not by PMA for
24 and 48 h. A, TNF mRNA were analyzed at
t = 72 h (just before RA treatment) and at the
indicated times (0-24 h) after PMA treatment (20 nM). The
TNF mRNA level in untreated cells is undetectable throughout the
duration of the experiment (not shown). This figure is representative
of two separate experiments; B, TNF antigen accumulation
in the culture medium measured at 24 and 48 h. Values are
mean ± S.D. (n = 6, three separate experiments
performed in duplicate). Values of control are significantly different
at p < 0.01 versus RA + PMA.
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We then evaluated the synthesis of PAI-1 in HL-525 cells under various
conditions. The PAI-1 mRNA level was hardly detectable in control
HL-525 cells as well as in cells treated by RA alone for 72 h or
longer (not shown). As shown in Fig.
7A, PMA alone has also no
significant effect on the PAI-1 mRNA level, whereas the RA + PMA
treatment resulted in a modest increase, which appeared 3 h after
PMA addition and continued over 9 h to slightly decrease at
24 h. The accumulation of PAI-1 antigen that was not increased by
either PMA or RA alone significantly increased in RA + PMA-treated HL-525 cells confirming previous data (22). Anti-TNF reduced the
level of PAI-1 mRNA in RA + PMA-treated HL-525 cells but, the
inhibitory effect could not be firmly detected before 9 h (Fig.
7A). In HL-525 cells treated with PMA alone, rhTNF did not significantly alter PAI-1 synthesis, both at mRNA and protein levels (not shown). However, when rhTNF was added to RA + PMA-treated HL-525 cells, a rapid and strong enhancement of the PAI-1
mRNA level was observed at 3 h and continued to increase over
24 h. This enhancement resulted in an increase in PAI-1 antigen
accumulation by a factor of 3 at 24 h and of 6.5 at 48 h
(Fig. 7B) when compared with RA + PMA-treated HL-525 cells.
Unlike native rhTNF , boiled rhTNF was ineffective. Fig. 3,
E-H shows that HL-525 cells treated by the combination of
RA + PMA develop a marked adhesion with some spreading and cell
extensions. These morphological patterns, particularly cell extensions,
were strongly intensified when rhTNF was added.

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Fig. 7.
Time-dependent analysis of PAI-1
synthesis in HL-525 cells. HL-525 cells were treated as in Fig. 5.
rhTNF (20 units/ml) or anti-TNF (10 µg/ml) were added
simultaneously with PMA (20 nM). A, PAI-1
mRNA were analyzed at indicated times corresponding to PMA
treatment. PAI-1 mRNA expression of untreated HL-525 cells or cells
treated with RA throughout the experiment (not shown) were identical to
those of untreated cells shown at t = 0. B,
PAI-1 antigen accumulation in the culture medium measured at 24 and
48 h. Values are mean ± S.D. (n = 6, three
separate experiments performed in duplicate). Values of control are
significantly different at p < 0.001 versus
RA + PMA, RA + PMA + TNF .
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Relationships between TNF and PAI-1 Synthesis in the Presence of
Other Inducers of Differentiation--
We evaluated if the autocrine
and stimulating effect of TNF was specific to PMA-induced
differentiation or also occurs with other agents known to trigger the
monocytic differentiation program. The treatment of the promonocytic
cell line U937 with rhTGF- 1/D3 for 24 h has been described to
induce monocytic differentiation (41). We treated HL-60 cells for
24 h with this mixture. The cells were then either stimulated or
not by rhTNF (t = 0). PAI-1 and TNF mRNA
levels were analyzed at 0, 5, and 8 h after stimulation (Fig.
8A). The combination of
rhTGF- 1/D3 alone increased the level of PAI-1 mRNA but not that
of TNF mRNA. The level of TNF secreted in the culture medium
was not different from that of nontreated cells (not shown).
Interestingly, in rhTGF- 1/D3-treated HL-60 cells, rhTNF enhanced
the level of PAI-1 mRNA (Fig. 8A). Consistent with PAI-1
mRNA levels, PAI-1 antigen accumulation was increased by
rhTGF- 1/D3 and was drastically potentiated by a factor of 4 after
the addition of rhTNF (Fig. 8B). In cells treated with
rhTGF- 1/D3, the percentage of adherent cells did not exceed 10-15%
at 24 h, but increased up to 50% when rhTNF was added.
However, adherence is less firm than with PMA, because cells can be
detached by shaking. We then investigated if PKC and MAPKp42/p44 were
involved in the TGF- 1/D3-induced PAI-1 synthesis, as we described
with PMA (22). Neither Ro 31-8220 (0.5 µM), a PKC
inhibitor, nor PD 098059 (10 µM), a MAPKK inhibitor,
altered the level of PAI-1 antigen in the culture medium of
rhTGF- 1/D3-treated cells (not shown). This result indicates that the
induction of differentiation by rhTGF- 1/D3 involves a signaling
pathway different from that induced by PMA.

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Fig. 8.
Time-dependent synthesis of PAI-1
and TNF in HL-60 cells treated by
rhTGF- 1/D3 mixture. HL-60 cells were
treated with a mixture of rhTGF- 1/D3 (10 ng/ml and 100 nmol/ml,
respectively) for 24 h. Then rhTNF (20 units/ml) was added
(t = 0) or not. At t = 0, rhTGF 1/D3-induced accumulation of PAI-1 antigen was of 12.4 ± 2.3 ng/106 cells. A, PAI-1 and TNF mRNAs
analyzed at the indicated times; B, PAI-1 antigen
accumulation in the culture medium measured 24 h after TNF
addition. Values are mean ± S.D. (n = 6, three
separate experiments performed in duplicate). Values of
rhTGF- 1/D3-treated cells are significantly different at
p < 0.01 versus rhTGF- 1/D3 + TNF .
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 |
DISCUSSION |
TNF is produced and released by activated macrophages in the
atherosclerotic lesion and is a major inducer of PAI-1 synthesis in
differentiated cells. However, its effect on PAI-1 in leukocytes appears linked to their differentiated stage because 1) TNF is inactive on monocytic progenitors (22, 29) and 2) mature macrophages produce more PAI-1 than monocytes isolated from circulation (7, 8). To
gain insight on this aspect, we used the undifferentiated human HL-60
leukemia cells that, when treated with differentiative agents, acquire
the monocyte/macrophage phenotype (19) and synthesize PAI-1. During
monocytic differentiation, PAI-1 gene expression depends on the early
activation (5 min) of the PKC /MAPKK/MAPKp42 pathway (22). Herein, we
show that an excess of anti-TNF dramatically reduced the PMA-induced
synthesis of PAI-1, observed at the mRNA level, 4 h after PMA
stimulation. This result strongly suggests that PMA-induced PAI-1
synthesis is in large part dependent on the autocrine action of
synthesized TNF . The modest up-modulating effect of rhTNF on
PAI-1 synthesis does not seem to result from a down-regulation of
TNF receptor surface expression, because it modestly increased after
PMA addition, although lesser than in control cells. Therefore, the
occupation of these receptors by endogenously released TNF likely
prevents rhTNF to strongly up-regulate PAI-1 synthesis. Because the
increase in the TNF mRNA level preceded that of PAI-1, one may
address the question if TNF is absolutely indispensable to initiate
PAI-1 synthesis. In the differentiation-resistant HL-525 cells cloned
from HL-60 cells (32, 39), RA + PMA treatment, which restores the
PKC -dependent Raf-1-MAPKK-MAPK pathway (42), increased
PAI-1 mRNA levels before those of TNF which plateaued at 10 h. Consequently, anti-TNF is effective much later than in HL-60
cells (9 versus 4 h) to inhibit PAI-1 synthesis. Thus,
the increase in PAI-1 mRNA level observed during the first 9 h
in RA + PMA-treated HL-525 cells is mainly the result of the
restoration of the PKC- -dependent pathway. Also, in
HL-60 cells a large excess of anti-TNF did not fully inhibit the
PMA-induced PAI-1 synthesis, suggesting that TNF is not an
obligatory preliminary step to initiate PAI-1 synthesis. As these data
supported the idea that the critical role of TNF is not to initiate
PAI-1 synthesis, we therefore addressed the question as to whether
TNF up-regulates it once the differentiation process is committed.
Our results indicate that this is the case.
First, in PMA-treated HL-525 cells, rhTNF that did not activate
PAI-1 synthesis strongly potentiates it when re-induction of
differentiation by RA was allowed. However, it should be noted that in
RA + PMA-treated HL-525 cells PAI-1 synthesis is lower than in HL-60
cells (22), although TNF is produced at levels comparable to those
produced by HL-60 cells. The reasons for this low efficiency are not
clear at present. A functional alteration of TNF receptors appears
unlikely, because the addition of rhTNF recovered the elevated rate
of PAI-1 synthesis. It is however, noteworthy that the optimum level of
TNF mRNA appeared much later than in HL-60 cells (10 versus 2 h) and obviously after that of PAI-1 mRNA.
Therefore, it is possible that in HL-525 cells, a time-dependent uncoupling between early
PKC -MAPKK-MAPKp42-induced PAI-1 gene activation and delayed action
of TNF prevents optimal up-regulation of PAI-1 gene expression. This
defect was corrected by the early addition of rhTNF , which can bind
to TNF receptors. A poor recovery in HL-525 cells treated by RA + PMA was observed for other phenotypes than that of PAI-1, such as
adherence and matrix metalloproteinase-9 production (31, 39). This
partial deficit can now at least be explained by a noncomplete
autocrine action of synthesized TNF .
Second, in HL-60 cells, the mixture of rhTGF- 1/D3 was described to
differentiate immature progenitors along the monocyte pathway (41).
With this combination, the level of PAI-1 synthesis at 24 h is
much lower than with PMA alone. As shown, this is because endogenous
synthesis of TNF is not activated by the combination of
rhTGF- 1/D3. Accordingly, the amplitude of up-regulation of PAI-1
synthesis provoked by rhTNF was much higher than with PMA. The
absence of significant TNF synthesis is probably related to the
signaling pathway triggered by TGF- 1/D3, which is different from
that induced by PMA. As proof, we demonstrated herein that PKC and MAPK
p42/p44 were not involved in rhTGF- 1/D3-induced PAI-1 synthesis
unlike we previously described for the early steps of PMA-induced PAI-1
synthesis (22). Recently, TFE3 and Smad proteins were shown to be
involved in TGF- 1-induced PAI-1 gene transcription (43, 44). Whether
these proteins can be up-regulated by TNF -mediated pathway remains
to be verified.
Third, the differentiation-dependent up-regulating effect
of TNF on PAI-1 synthesis is further demonstrated in PdBu-treated HL-60 cells. We showed that 14-20 h of the differentiation process is
necessary for TNF to exert its optimal potentiating effect.
Our present results can be compared with those we obtained in
promonocytic U937 cells (27) in which TNF potentiated PAI-1 synthesis initially activated by thapsigargin, a non-PMA-type differentiative agent. Taken as a whole, these results indicate that
whatever the type of differentiative agents inducing expression of
factors involved in PAI-1 gene transcription, these factors are further
activated by endogenous TNF which in turn up-regulates PAI-1
synthesis. It is clear that the amplitude of the autocrine TNF
effect is dependent on the potency of differentiative agents to induce
TNF synthesis.
The up-regulation of PMA-induced PAI-1 synthesis by TNF is
consistent with the fact that TNF stimulates AP-1 activity through a
prolonged activation of c-Jun NH2-terminal kinase (45). In PMA- or vitamin D3-differentiated HL-60 cells, an increase in c-jun mRNA expression is observed (46). Interestingly,
homodimers of c-Jun were shown to bind to the tetradecanoyl phorbol
acetate-response element at position 58 to 50 of the PAI-1 promoter
in HepG2 stimulated by PMA (23). Also, in human fibroblasts, the
immediate-early gene egr-1 was recently demonstrated to be
induced by TNF (47) via the c-Jun NH2-terminal kinase
pathway (48), and in the HT-1080 fibrosarcoma cell line,
egr-1 enhanced synthesis of PAI-1 (18). We reported that
SB203580 (22), a potent specific inhibitor of the stress-activated
kinase p38 (49), and Emodin,2
a potent inhibitor of NF- B activation (50), have no effect on
PMA-induced PAI-1 synthesis. Collectively, these data are in favor of
the involvement of c-Jun NH2-terminal kinase in the TNF signaling pathway leading to PAI-1 up-regulation, which is presently under investigation.
The functional importance of TNF and likely also PAI-1 is underlined
here with the morphological aspect of adherent differentiated cells
treated by rhTNF . Recently, it was shown that HL-60 cells treated
with PMA secrete fibronectin and express its respective receptor, the
integrin 5 1 (31, 51). This receptor is
induced by TNF in an autocrine manner and leads to the secretion of
the 92-kDa gelatinase involved in tissue remodeling. The comparable autocrine regulation by TNF of PAI-1 synthesis poses the question of
the role of this inhibitor in participating to pericellular proteolysis
during leukocyte differentiation. Interestingly, it was recently shown
in the HT-1080 cell line that the induction of PAI-1 driven by TGF- 1
via Egr-1 stimulation is coordinated with the secretion of fibronectin
and its respective receptors, including a role for PAI-1 to stabilize
cell attachment (18). Recent experimental data support the contention
that PAI-1 is specifically directed at sites where pericellular
proteolysis must be controlled (52). In cultured vascular smooth muscle cells, PAI-1 limits plasmin-mediated matrix metalloproteinase activation and consequently may prevent an excessive matrix proteolysis (53).
In conclusion, our results indicate that during induction of
monocyte/macrophage differentiation, synthesized TNF up-regulates in
an autocrine manner the PAI-1 synthesis, provided the PAI-1 gene has
been primed by the differentiative stimulus.
 |
ACKNOWLEDGEMENTS |
We are indebted to E. Huberman (Argonne, IL)
for providing HL-525 cells, P. Declerck and R. Lijnen (Leuven, Belgium)
for providing monoclonal antibodies directed against PAI-1, M. Verdier
for PAI-1 assays, N. Fernandez for flow cytometry, and V. Thomé
for preparing cell cultures.
 |
FOOTNOTES |
*
This work was supported by funds of INSERM and
Université de la Méditerranée.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Recipient of funds from Groupe d'Etudes en Hémostase et
Thrombose-Sanofi and from the Fondation pour la Recherche
Médicale.
§
To whom correspondence should be addressed: EPI 99-36, Laboratoire
d'Hématologie, Faculté de Médecine, 27 Bd. Jean
Moulin, 13385 Marseille Cedex 5, France. Tel.: (33) 4 91 32 45 07; Fax: (33) 4 91 25 43 36; E-mail: Gilles.Nalbone@medecine.univ-mrs.fr.
2
S. Lopez and G. Nalbone, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
PAI-1, plasminogen
activator inhibitor type-1;
PKC, protein kinase C;
MAPK, mitogen-activated protein kinase;
MAPKK, mitogen-activated protein
kinase kinase;
PMA, phorbol 12-myristate 13-acetate;
TNF , tumor
necrosis factor ;
PdBu, phorbol dibutyrate;
rh, recombinant human;
TGF, transforming growth factor;
RT, reverse transcriptase;
RA, retinoic acid;
D3, 1 ,25-dihydroxyvitamin D3;
PCR, polymerase chain
reaction;
bp, base pairs;
eEF1 , eukaryotic elongation factor
1 .
 |
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