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J Biol Chem, Vol. 275, Issue 5, 3081-3087, February 4, 2000


Tumor Necrosis Factor alpha  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 LopezDagger , 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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tumor necrosis factor-alpha (TNFalpha ) 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 TNFalpha contributed to plasminogen activator inhibitor type-1 (PAI-1) synthesis that is initiated by a protein kinase Cbeta -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 TNFalpha mRNA strongly increased and appeared earlier than PAI-1 mRNA. An anti-TNFalpha antibody significantly inhibited the PMA-induced PAI-1 mRNA and protein levels. The recombinant human TNFalpha , 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 factorbeta -1/vitamin D3 differentiative mixture confirmed the relationships between the induction of differentiation and the potency of TNFalpha to up-regulate PAI-1 synthesis. In conclusion, we showed that during the induction of monocyte/macrophage differentiation, TNFalpha and PAI-1 gene expressions are activated and that synthesized TNFalpha up-regulates and prolongs, in an autocrine manner, the synthesis of PAI-1.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha vbeta 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 PKCbeta -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). TNFalpha , 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 TNFalpha (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 TNFalpha (30), which was recently demonstrated to up-regulate, in an autocrine manner, the synthesis of 92-kDa gelatinase via alpha 5beta 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 TNFalpha 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, TNFalpha and PAI-1 mRNA levels increased. The synthesized TNFalpha up-regulates and prolongs, in an autocrine manner, elevated PAI-1 mRNA levels resulting in enhanced protein antigen synthesis and secretion.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 TNFalpha were from R&D Systems. Recombinant human TGF-beta 1 (rhTGF-beta 1) and TNFalpha (rhTNFalpha ) 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 1alpha ,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 TNFalpha (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 (eEF1alpha ) was observed to be a stable house keeping gene, whereas in TGF-beta 1/D3-treated HL-60 cells, beta -actin was preferred. The amplified fragment for eEF1alpha (GenBankTM accession number X03558) is 289 bp, base position 382-670 and is 388 bp for beta -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 TNFalpha and its receptors, PAI-1, and beta -actin cDNAs and a number of 18 cycles for eEF1alpha . 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, eEF1alpha , and beta -actin) or 57 °C (for TNFalpha 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). TNFalpha 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 TNFalpha 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 TNFalpha . 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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PMA Increases TNFalpha mRNA Level Earlier than That of PAI-1

In nonstimulated HL-60 cells, the level of TNFalpha mRNA was low (Fig. 1A). When HL-60 cells were treated with 20 nM PMA, the TNFalpha mRNA level rose rapidly. The optimum expression was attained 2 h after PMA addition. The increase in TNFalpha mRNA level was transient since 24 h after PMA addition, the level was below that of nonstimulated cells (Fig. 1A). TNFalpha 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 TNFalpha 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 TNFalpha 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 TNFalpha 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.

Endogenous TNFalpha Up-regulates PMA-induced PAI-1 Synthesis

In view of the above results and because PAI-1 is a TNFalpha -responsive gene in various differentiated cells, we then examined if the synthesis of PAI-1 was attributable only to the activation of the PKCbeta -MAPKK-MAPKp42 pathway (22) or if the released TNFalpha contributed to the synthesis of PAI-1. HL-60 cells were simultaneously incubated with a neutralizing antibody against human TNFalpha and PMA. Preliminary experiments revealed that the optimal effect was obtained at 2.5 µg/ml anti-TNFalpha . We used 10 µg/ml to ensure complete quenching of released TNFalpha . As shown in Fig. 2A, anti-TNFalpha significantly lowered the level of PAI-1 mRNA 4 h after PMA addition indicating that endogenously released TNFalpha 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-TNFalpha 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 TNFalpha (34). The IgG-induced TNFalpha 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-TNFalpha 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-TNFalpha was added. These results indicate that, in PMA-stimulated HL-60 cells, endogenous TNFalpha up-regulates, in an autocrine manner, the PAI-1 synthesis.


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  		Fig. 2.   Time-dependent effects of anti-TNFalpha and rhTNFalpha 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-TNFalpha (10 µg/ml) or rhTNFalpha (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 + TNFalpha and p < 0.01 versus PMA + anti-TNFalpha .


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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-TNFalpha ; D, PMA + rhTNFalpha . Lower, HL-525 cells; E, nontreated; F, PMA + rhTNFalpha ; G, RA + PMA; H, RA + PMA + rhTNFalpha . Morphology of HL-525 cells treated by RA, rhTNFalpha , or RA + rhTNFalpha (not shown) are similar to that of nontreated cells. Magnification × 40.

The Up-regulating Effect of TNFalpha on PAI-1 Synthesis Is Linked to the Induction of Differentiation

rhTNFalpha Activates PAI-1 Synthesis in PMA-treated HL-60 Cells-- rhTNFalpha 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 rhTNFalpha 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, rhTNFalpha 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 rhTNFalpha 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 rhTNFalpha 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 TNFalpha 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).

The lack of effect of rhTNFalpha on PAI-1 synthesis observed on native undifferentiated HL-60 cells is not linked to some defect in TNFalpha 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 TNFalpha to activate PAI-1 gene expression. We thus evaluated the relationship between the time-dependent monocyte/macrophage differentiation and the potency of TNFalpha to activate PAI-1 synthesis. To specifically evaluate the effect of rhTNFalpha 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 rhTNFalpha for 24 h. As shown in Fig. 5, the enhancing effect of rhTNFalpha 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 rhTNFalpha 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 rhTNFalpha activates PAI-1 synthesis once HL-60 cells have been "primed" by a differentiative stimulus.


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  		Fig. 5.   Effect of rhTNFalpha 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. rhTNFalpha (20 units/ml) was added (hatched bar) or not (gray bar). PAI-1 was measured in the culture medium 24 h after rhTNFalpha addition. Values are mean ± S.D. (n = 4, two separate experiments performed in duplicate).

Relationships between TNFalpha 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 PKCbeta 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 PKCbeta gene expression (40).

Fig. 6A shows that, in HL-525 cells, the level of TNFalpha 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 TNFalpha mRNA levels, except a slight transient increase at 6-8 h. Clearly, RA pretreatment allows PMA to progressively and strongly enhance TNFalpha mRNA levels. It should be noticed that the optimum level of TNFalpha mRNA significantly increased much later than in HL-60 cells (10 versus 2 h). The amount of TNFalpha antigen secreted into the culture medium (Fig. 6B) showed that, in comparison with control HL-525 cells, PMA modestly enhanced TNFalpha antigen accumulation, whereas RA alone had no marked effect. The combination of RA + PMA increased the antigen level of TNFalpha 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 TNFalpha 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, TNFalpha 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 TNFalpha mRNA level in untreated cells is undetectable throughout the duration of the experiment (not shown). This figure is representative of two separate experiments; B, TNFalpha 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.

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-TNFalpha 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, rhTNFalpha did not significantly alter PAI-1 synthesis, both at mRNA and protein levels (not shown). However, when rhTNFalpha 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 rhTNFalpha , boiled rhTNFalpha 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 rhTNFalpha 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. rhTNFalpha (20 units/ml) or anti-TNFalpha (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 + TNFalpha .

Relationships between TNFalpha and PAI-1 Synthesis in the Presence of Other Inducers of Differentiation-- We evaluated if the autocrine and stimulating effect of TNFalpha 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-beta 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 rhTNFalpha (t = 0). PAI-1 and TNFalpha mRNA levels were analyzed at 0, 5, and 8 h after stimulation (Fig. 8A). The combination of rhTGF-beta 1/D3 alone increased the level of PAI-1 mRNA but not that of TNFalpha mRNA. The level of TNFalpha secreted in the culture medium was not different from that of nontreated cells (not shown). Interestingly, in rhTGF-beta 1/D3-treated HL-60 cells, rhTNFalpha enhanced the level of PAI-1 mRNA (Fig. 8A). Consistent with PAI-1 mRNA levels, PAI-1 antigen accumulation was increased by rhTGF-beta 1/D3 and was drastically potentiated by a factor of 4 after the addition of rhTNFalpha (Fig. 8B). In cells treated with rhTGF-beta 1/D3, the percentage of adherent cells did not exceed 10-15% at 24 h, but increased up to 50% when rhTNFalpha 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-beta 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-beta 1/D3-treated cells (not shown). This result indicates that the induction of differentiation by rhTGF-beta 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 TNFalpha in HL-60 cells treated by rhTGF-beta 1/D3 mixture. HL-60 cells were treated with a mixture of rhTGF-beta 1/D3 (10 ng/ml and 100 nmol/ml, respectively) for 24 h. Then rhTNFalpha (20 units/ml) was added (t = 0) or not. At t = 0, rhTGFbeta 1/D3-induced accumulation of PAI-1 antigen was of 12.4 ± 2.3 ng/106 cells. A, PAI-1 and TNFalpha mRNAs analyzed at the indicated times; B, PAI-1 antigen accumulation in the culture medium measured 24 h after TNFalpha addition. Values are mean ± S.D. (n = 6, three separate experiments performed in duplicate). Values of rhTGF-beta 1/D3-treated cells are significantly different at p < 0.01 versus rhTGF-beta 1/D3 + TNFalpha .


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

TNFalpha 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) TNFalpha 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 PKCbeta /MAPKK/MAPKp42 pathway (22). Herein, we show that an excess of anti-TNFalpha 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 TNFalpha . The modest up-modulating effect of rhTNFalpha on PAI-1 synthesis does not seem to result from a down-regulation of TNFalpha 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 TNFalpha likely prevents rhTNFalpha to strongly up-regulate PAI-1 synthesis. Because the increase in the TNFalpha mRNA level preceded that of PAI-1, one may address the question if TNFalpha 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 PKCbeta -dependent Raf-1-MAPKK-MAPK pathway (42), increased PAI-1 mRNA levels before those of TNFalpha which plateaued at 10 h. Consequently, anti-TNFalpha 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-beta -dependent pathway. Also, in HL-60 cells a large excess of anti-TNFalpha did not fully inhibit the PMA-induced PAI-1 synthesis, suggesting that TNFalpha is not an obligatory preliminary step to initiate PAI-1 synthesis. As these data supported the idea that the critical role of TNFalpha is not to initiate PAI-1 synthesis, we therefore addressed the question as to whether TNFalpha up-regulates it once the differentiation process is committed. Our results indicate that this is the case.

First, in PMA-treated HL-525 cells, rhTNFalpha 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 TNFalpha 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 TNFalpha receptors appears unlikely, because the addition of rhTNFalpha recovered the elevated rate of PAI-1 synthesis. It is however, noteworthy that the optimum level of TNFalpha 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 PKCbeta -MAPKK-MAPKp42-induced PAI-1 gene activation and delayed action of TNFalpha prevents optimal up-regulation of PAI-1 gene expression. This defect was corrected by the early addition of rhTNFalpha , which can bind to TNFalpha 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 TNFalpha .

Second, in HL-60 cells, the mixture of rhTGF-beta 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 TNFalpha is not activated by the combination of rhTGF-beta 1/D3. Accordingly, the amplitude of up-regulation of PAI-1 synthesis provoked by rhTNFalpha was much higher than with PMA. The absence of significant TNFalpha synthesis is probably related to the signaling pathway triggered by TGF-beta 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-beta 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-beta 1-induced PAI-1 gene transcription (43, 44). Whether these proteins can be up-regulated by TNFalpha -mediated pathway remains to be verified.

Third, the differentiation-dependent up-regulating effect of TNFalpha 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 TNFalpha to exert its optimal potentiating effect.

Our present results can be compared with those we obtained in promonocytic U937 cells (27) in which TNFalpha 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 TNFalpha which in turn up-regulates PAI-1 synthesis. It is clear that the amplitude of the autocrine TNFalpha effect is dependent on the potency of differentiative agents to induce TNFalpha synthesis.

The up-regulation of PMA-induced PAI-1 synthesis by TNFalpha is consistent with the fact that TNFalpha 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 TNFalpha (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-kappa 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 TNFalpha signaling pathway leading to PAI-1 up-regulation, which is presently under investigation.

The functional importance of TNFalpha and likely also PAI-1 is underlined here with the morphological aspect of adherent differentiated cells treated by rhTNFalpha . Recently, it was shown that HL-60 cells treated with PMA secrete fibronectin and express its respective receptor, the integrin alpha 5beta 1 (31, 51). This receptor is induced by TNFalpha in an autocrine manner and leads to the secretion of the 92-kDa gelatinase involved in tissue remodeling. The comparable autocrine regulation by TNFalpha 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-beta 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 TNFalpha 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.

Dagger 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; TNFalpha , tumor necrosis factor alpha ; PdBu, phorbol dibutyrate; rh, recombinant human; TGF, transforming growth factor; RT, reverse transcriptase; RA, retinoic acid; D3, 1alpha ,25-dihydroxyvitamin D3; PCR, polymerase chain reaction; bp, base pairs; eEF1alpha , eukaryotic elongation factor 1alpha .

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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