From INSERM U364 and § Groupe de Recherche
en Immunopathologie de la Leishmaniose, Laboratoire de Parasitologie,
Faculté de Médecine Pasteur, Avenue de Valombrose,
06107 Nice Cedex 02, France
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ABSTRACT |
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We have demonstrated previously that microtubule depolymerization by colchicine in human monocytes induces selective production of interleukin-1 (IL-1) (Manié, S., Schmid-Alliana, A., Kubar, J., Ferrua, B., and Rossi, B. (1993) J. Biol. Chem. 268, 13675-13681). Here, we provide evidence that disruption of the microtubule structure rapidly triggers extracellular signal-regulated kinase (ERK) activation, whereas it was without effect on SAPK2 activity, which is commonly acknowledged to control pro-inflammatory cytokine production. This process involves the activation of the entire cascade including Ras, Raf-1, MEK1/2, ERK1, and ERK2. Activation of ERKs is followed by their nuclear translocation. Although other SAPK congeners might be activated upon microtubule depolymerization, the activation of ERK1 and ERK2 is mandatory for IL-1 production as shown by the blocking effect of PD 98059, a specific MEK1/2 inhibitor. Additionally, we provide evidence that microtubule disruption also induces the activation of c-Src and Hck activities. The importance of Src kinases in the mediation of the colchicine effect is underscored by the fact that CP 118556, a specific inhibitor of Src-like kinase, abrogates both the colchicine-induced ERK activation and IL-1 production. This is the first evidence that ERK activation is an absolute prerequisite for induction of this cytokine. Altogether, our data lend support to a model where the status of microtubule integrity controls the level of Src activities that subsequently activate the ERK kinase cascade, thus leading to IL-1 production.
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INTRODUCTION |
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Monocytes play an essential role in the inflammation as accessory
cells for the processing and presentation of antigen to lymphocytes,
but also in the inflammatory process by releasing oxygen metabolites,
lysosomal enzymes, and cytokines such as tumor necrosis factor- and
interleukins (ILs)1 1 and 6. The regulation of the synthesis of these cytokines is complex, and
still poorly understood. Bacterial endotoxin (lipopolysaccharides), antigen-antibody complexes, phorbol esters (phorbol 12-myristate 13-acetate), and cytokines are classical monocyte-activating agents which induce the concerted production of these pro-inflammatory cytokines. Recent studies have shown that alteration of the
cytoskeletal network by chemical agents also induces a dramatic and
specific increase of IL-1 (1, 2) and tumor necrosis factor-
(3, 4)
by monocytic cells.
During recent years, growing evidence has accumulated showing that the
cytoskeleton could intervene in the propagation of the mitogenic and
activation signal (5). In this respect, many studies have focused on
the actin network that acts as a dynamic structure involved in the
integrin-mediated signaling cascade (6). Cytoplasmic microtubules
represent another major element of the cytoskeleton that have been
implicated in diverse processes such as cellular motility,
intracellular transport, and secretion. The fact that microtubules are
subject to constant remodeling, because of the dynamic instability of
tubulin dimers, prompted us to consider that the microtubule network
may be an important actor in the transmission of activation signals
inside the cell. This idea is supported by reports showing that: (i)
microtubule reorganization, occurring during differentiation of HL 60 cells, is associated to tubulin phosphorylation on tyrosine residues (7); (ii) microtubule reorganization were also observed after cytokines
and phorbol ester treatment of human umbilical vein endothelial cells
(8); and (iii) microtubule disruption generates a signal that leads to
NFB activation (9).
We and others have shown that microtubule-disrupting drugs are capable of generating a signal that leads to the selective induction of IL-1 synthesis in human monocytes (1, 2). We demonstrate that microtubule depolymerization was without effect on SAPK2 activity, which is generally considered as a key regulating enzyme in the production of cytokines (10, 11). In contrast, we provide the first evidence that the colchicine-mediated microtubule disassembly can induce, by itself, the activation of the entire Ras-dependent cascade leading to ERK activation and their translocation to the nucleus. This cascade is in fact triggered by the upstream transient activation of c-Src and Hck. Owing to the selective effect of colchicine on IL-1 production, this study emphasizes the importance of the microtubule polymerization state in the control of the regulation it exerts on some Src-like kinases.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and Human Monocyte Preparation
Human myelomonocytic THP1 cells (ATCC) were grown in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal calf serum (Biosepra), 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (referred to as the complete medium). Fetal calf serum was tested for the absence of endotoxin (<0.1 IU/ml, Institut J. Boy, Reims, France). Cells were maintained at 37 °C in a humidified 5% CO2 atmosphere.
Human peripheral blood mononuclear cells were isolated under sterile conditions from leukophoresis samples obtained from the Center de Transfusion Sanguine A. Tzanck (St. Laurent du Var, France) and treated as reported previously (1). Monocytes were cultured for 48 h to allow adherence-induced transcription of IL-1 mRNA to subside.
Measurement of IL-1 Production
Human monocytes (5 × 105 cells/ml) were
stimulated for 18 h in 0.5 ml of complete medium (48-well plates,
Nunc) in the presence of effectors. IL-1 production (cell-associated
and secreted forms) was assayed in the cell culture medium by using a
specific IL-1
sandwich enzyme-linked immunosorbent assay as
described previously (12).
Nuclei Isolation and Run-on Transcription Assay
Human monocytes or THP1 cells (7 × 105 cells/ml) were starved 16 h in RPMI 1640 medium and then harvested by a 5-min centrifugation at 1000 × g before being resuspended in RPMI-Hepes (Life Technologies, Inc.) at a concentration of 2 × 107 cells/ml. Cells (5 × 107) were treated with or without the effectors for 3 h at 37 °C. Nuclei were isolated from cells, and the run-on transcription assay was performed as described (13).
Products
Colchicine and lumicolchicine were obtained from Sigma. Genistein and herbimycin A were from Life Technologies, Inc. PD 98059, the microtubule-associated protein (MAP) kinase kinase (MEK) inhibitor, was purchased from New England Biolabs (Beverly, MA). CP 118556 (also named PP1) was kindly provided by S. Kadin (Pfizer Research, Groton, CT). SB 203580 was a gift from SmithKline Beecham Pharmaceuticals (King of Prussia, PA). Pervanadate was prepared as described previously (14).
Cell Stimulation and Cell Lysis
Human monocytes or THP1 cells (7 × 105 cells/ml) were starved 16 h in RPMI 1640 medium and harvested by centrifugation for 5 min at 1000 g before being resuspended in RPMI-Hepes (Life Technologies, Inc.) at a concentration of 2 × 107 cells/ml. Cells (107) were treated with or without the effectors for the indicated times at 37 °C and lysed in buffer (150 mM NaCl, 0.8 mM MgCl2, 5 mM EGTA, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 15 µg/ml leupeptin, 1 µM pepstatin, 1 mM Na3VO4, and 50 mM Hepes, pH 7.5). The crude lysates were centrifuged at 18,000 × g for 20 min at 4 °C, and the supernatants were precleared with nonimmune serum prebound to protein A-Sepharose (Pharmacia Biotech Inc.) for rabbit serum or protein G-Sepharose (Santa Cruz Biotechnology, Santa Cruz, CA) for sheep serum. The precleared lysates were incubated at 4 °C for 16 h with antibodies raised against the various transduction proteins previously bound to protein A-Sepharose or to protein G-Sepharose. All antibodies were used at dilution 1/100.
Immune Complex Kinase Assay
Src Kinase Activity--
The Src-related kinases were
immunoprecipitated with anti-c-Src antibodies (Santa Cruz
Biotechnology) bound to protein G-Sepharose or with anti-Hck antibodies
(gift from I. Maridonneau-Parini) bound to protein A-Sepharose. The
immunopellets were washed twice with lysis buffer, followed by one wash
with lysis buffer supplemented with 0.25% deoxycholate, and ultimately
washed twice in Src kinase buffer (5 mM MgCl2,
5 mM MnCl2, 30 mM Hepes, pH 7.5).
Samples were then resuspended in 50 µl of Src kinase buffer
supplemented with 0.1 mg/ml acid-denatured enolase, which was used as
an exogenous substrate. The kinase assay was started by addition of 40 µCi/ml [-32P]ATP (370 MBq/ml, Amersham Life
Science). After addition of 25 µl of 9 × Laemmli sample buffer
to stop the reaction, samples were heated to 95 °C for 3 min and
analyzed by SDS-PAGE on a 10% gel under reducing conditions. Gels were
exposed for autoradiography on Hyperfilm (Amersham).
MAP Kinase Activity--
MAP-related kinases were
immunoprecipitated with anti-SAPK2/P38, anti-ERK1, or anti-ERK2
antisera (Santa Cruz Biotechnology) bound to protein A-Sepharose.
Immunopellets were washed twice with lysis buffer, twice with MAP
kinase buffer (30 mM NaCl, 0.1% Nonidet P-40, 10%
glycerol, 200 µM Na3VO4, 30 mM Hepes, pH 7.5), and resuspended in 50 µl of MAP kinase
buffer in the presence of 75 µM ATP, 30 mM Mg
acetate, and 0.2 mg/ml myelin basic protein (MBP, Sigma), which was
used as an exogenous substrate. The kinase assay was initiated by
addition of 100 µCi/ml [-32P]ATP. After addition of
25 µl of 9 × Laemmli sample buffer to stop the reaction, the
samples were heated to 95 °C for 3 min. Samples were split to be
analyzed by SDS-PAGE. Gels were either exposed for autoradiography
using hyperfilms (Amersham) or probed with antibodies for
immunoblotting experiments. Autoradiographies were scanned using a
Ultro-Scan laser densitometer.
MEK Kinase Activity--
MEK was immunoprecipitated from
colchicine-treated cells with anti-MEK-1 antibody (Santa Cruz
Biotechnology) bound to protein A-Sepharose. The immunopellets were
washed twice with lysis buffer and twice with MEK kinase buffer (1 mM MnCl2, 10 mM MgCl2,
1 mM dithiothreitol, 10 mM
p-nitrophenylphosphate, 30 mM Hepes, pH 7.5). In
parallel, using specific antibodies, we immunoprecipitated ERK1, from
unstimulated THP1 lysates, to be used as a substrate. The pellets
containing MEK and ERK1, respectively, were then mixed in a final
volume of 50 µl of MEK kinase buffer in the presence of 15 µM ATP. The reaction was started by addition of 100 µCi/ml [-32P]ATP. After addition of 25 µl of
9 × Laemmli sample buffer to stop the reaction, the samples were
heated to 95 °C for 3 min. Samples were split to be analyzed by
SDS-PAGE under reducing conditions, proteins were revealed either by
autoradiography or by Western blotting using appropriate
antibodies.
Raf-1 Kinase Activity--
Raf-1 was immunoprecipitated with
anti-c-Raf-1 antibody (Santa Cruz Biotechnology) bound to protein
A-Sepharose. The immunopellets were washed twice with lysis buffer and
twice with MEK kinase buffer. In parallel, MEK was immunoprecipated
from lysates of unstimulated THP1 with anti-MEK-1 antibodies to be used
as a Raf-1 substrate. The pellets containing Raf-1 or MEK were then
mixed to a final volume of 50 µl of MEK kinase buffer in the presence of 15 µM ATP. The reaction was started by addition of 100 µCi/ml [-32P]ATP and stopped by addition of 25 µl
of 9 × Laemmli sample buffer. Samples were heated to 95 °C for
3 min and split for analysis by autoradiography or Western blotting
after separation by SDS-PAGE under reducing conditions.
Western Blotting Analysis
Total cell lysates (100 µg), immunoprecipitated substrates, or nuclear proteins (100 µg) were separated by SDS-PAGE and transferred to Immobilon membrane as detailed previously (15). The blots were probed with 4G10 anti-phosphotyrosine (Upstate Biotechnology, Inc., Lake Placid, NY) or anti-c-Src at 1 µg/ml, or with anti-ERK1, anti-ERK2, anti-SAPK2/P38, anti-Raf-1, anti-MEK-1, or anti-Hck (Santa Cruz Biotechnology) at 0.1 µg/ml, the proteins were visualized by the Amersham ECL system and quantified by densitometric scanning.
Determination of Ras-GTP/Ras-GDP Ratio
THP1 cells (7 × 105 cells/ml) were starved for 16 h in RPMI 1640 medium and subsequently labeled for 3 h at a concentration of 2 × 107 cells/ml with 1 mCi of [32P]orthophosphate (200 mCi/mmol, Amersham) in phosphate-free buffer (140 mM NaCl, 5 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, 10 mM glucose, 20 mM Hepes, pH 7.4). Cells (107) were stimulated or not by colchicine (1 µM) for the indicated times at 37 °C and rapidly washed with phosphate-free buffer, before being lysed in buffer (150 mM NaCl, 0.8 mM MgCl2, 5 mM EGTA, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 15 µg/ml leupeptin, 1 µM pepstatin, 1 mM Na3VO4, and 50 mM Hepes, pH 7.5). After 20 min, NaCl, SDS, and deoxycholate were added to a final concentration of 0.5 M, 0.5% and 0.05%, respectively. The crude lysates were centrifuged at 18 000 × g for 20 min at 4 °C and the supernatants were precleared by incubation with rat nonimmune serum bound to protein G-Sepharose for 30 min. The treated lysates were then incubated at 4 °C for 2 h with anti-p21ras prebound to protein G-Sepharose (Santa Cruz Biotechnology). The immunoprecipitates were collected and washed eight times with 50 mM Hepes buffer, pH 7.4, 500 mM NaCl, 5 mM MgCl2, 0.1% Nonidet P-40, 0.005% SDS. GTP and GDP were finally eluted in 5 mM dithiothreitol, 5 mM EDTA, 0.2% SDS, 0.5 mM GTP, and 0.5 mM GDP at 68 °C for 20 min and separated on polyethyleneimine-cellulose plates (Schleicher & Schüll), developed in 1.2 M ammonium formate, 0.8 M HCl. Plates were exposed for autoradiography, and the GTP/GDP ratio was determined by densitometric scanning.
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RESULTS |
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Colchicine Stimulates Pro-IL-1 mRNA Transcription in Human
Monocytes--
To elucidate the step at which colchicine controls the
induction of IL-1
mRNA expression (1), run-on experiments were carried out. Nascent nuclear RNA chains, biosynthetically labeled with
[
-32P]UTP, were isolated from human monocytes
previously stimulated for 3 h with colchicine or lumicolchicine.
Labeled RNAs were then hybridized to nitrocellulose filters previously
spotted with plasmids harboring either the IL-1
or the
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) coding sequence. A
detectable constitutive level of IL-1
transcription was observed
(Fig. 1, lane 1), but the
treatment with colchicine dramatically increased the IL-1
transcription rate (Fig. 1, lane 3). Under the same
conditions, lumicolchicine, the inactive analog of colchicine, was
without any effect (lane 2). The slight inhibition that
lumicolchicine induced on IL-1
transcription was not significant
when normalized against the corresponding GAPDH spot. These results
suggest that disruption of the microtubule network increased
pro-IL-1
mRNAs at the level of transcription.
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Microtubule Depolymerization Does Not Increase SAPK2/P38 Activity
but Does Stimulates the Kinase Activity of ERK1 and ERK2 and Causes
Their Nuclear Translocation--
It has been reported that
microtubule-disrupting agents, such as colchicine or
microtubule-stabilizing agents, e.g. taxol, could modulate
tyrosine kinase activities in intact cells (4, 16, 17). On the basis of
these observations, we hypothesized that tyrosine kinase activities
might participate in colchicine-induced IL-1 production. We first
tested the effects of herbimycin A and genistein, two potent tyrosine
kinase inhibitors, on the colchicine-induced IL-1
production in
human monocytes. As shown in Fig.
2A (upper panel),
herbimycin A and genistein both inhibited colchicine-induced IL-1
production, with ID50 values of 10 nM and 3 µM, respectively. Conversely, as shown in Fig.
2A (lower panel), the colchicine-induced IL-1
production was dramatically increased in the presence of pervanadate, a
potent inhibitor of tyrosine phosphatases (14) with a maximal
synergizing effect at 30 µM. Similar effects of herbimycin A, genistein, and pervanadate were obtained when the production of the IL-1
protein was assayed instead of IL-1
(data not shown). It is noteworthy that herbimycin A does not prevent colchicine-induced cAMP accumulation in human monocytes (data not
shown), suggesting that the colchicine-induced cAMP response, as
reported previously (18, 19), is regulated by a pathway distinct from
that controlled by the herbimycin A-sensitive tyrosine kinase
activity.
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Colchicine Activates MEK and Raf-1 Kinase and Stimulates Nucleotide Exchange on p21ras-- To understand the mechanisms by which microtubule depolymerization stimulated ERKs, we studied the colchicine effect on the upstream acting MEK and Raf-1 kinase activities.
It is well established that ERK1 and ERK2 are activated by the upstream MEK-1 and MEK-2 kinases (23, 24). The MEK-1 activity was measured in our system by mixing an anti-MEK-1 immunoprecipitate from colchicine-treated THP1 cell lysates with an immunopurified ERK1 inactive fraction obtained from unstimulated cell lysates. The data presented in Fig. 6A (upper panel) provide evidence that microtubule depolymerization (lanes 2-6) stimulates MEK-1 activity. Phosphorylation of ERK1 used as substrate rose steeply after 5 min of treatment, to culminate at 30 min, and then slowly declined by 60 min. It is noteworthy that the time course of ERK1 phosphorylation in response to colchicine treatment paralleled that of MEK-1 autophosphorylation (Fig. 6, lower panel).
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Microtubule Depolymerization Stimulates the Kinase Activity of c-Src and Hck in Human Monocytic Cells-- As noted above (Fig. 2), the stimulation of human monocytic cells by colchicine resulted in an increased tyrosine phosphorylation of a protein set, including proteins in the 55-60-kDa range, which are reminiscent of Src kinase congeners (31-33). We thus examined the ability of colchicine to stimulate the kinase activity of four members of the Src family that are expressed in THP1 cells, namely pp60c-src, pp59hck, pp53/56lyn, and pp59fyn. The four kinases were immunoprecipitated with appropriate antibodies from unstimulated or colchicine-treated THP1 cell lysates and then tested for their ability to phosphorylate the exogenous substrate enolase in vitro and to undergo auto-phosphorylation. As shown in Fig. 7 (A and B), c-Src as well as Hck activities peaked at 5-10 min and subsided within 30 min following colchicine treatment. The extent of autophosphorylation of the two kinases in response to microtubule depolymerization paralleled their respective kinase activity toward enolase. Interestingly, activation of c-Src and Hck did not reflect a general increase in Src kinase activity, since Lyn activity remained unaffected, while Fyn activity was slightly diminished by the colchicine treatment (data not shown).
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DISCUSSION |
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Several lines of evidence support the idea that changes in the
cell shape could trigger by itself a signal that leads to gene regulation (35). In this regard, microtubules, the architecture of
which undergoes a constant remodeling due to the dynamic instability of
tubulin dimers, appear as a good candidate, susceptible to rapidly
convert a structural change in a signaling pathway in response to
external signals. We and others have reported previously that
microtubule-disrupting agents can induce the synthesis and secretion of
IL-1 and IL-1
in human monocytes/macrophages (1, 2) at the
exclusion of the other inflammatory cytokines. This model provides thus
a valuable tool to unravel the signaling pathways that selectively
control IL-1 production.
In this study, we first ascertained by means of run-on experiments that
accumulation of the pro-IL-1 transcript, as evoked by microtubule
depolymerization, relied on an increased transcription rate of
pro-IL-1
mRNA. These data suggest that microtubule
depolymerization not only controlled events occurring in the
cytoplasmic compartment, but also conveyed a signal to the nucleus
resulting in the activation of transcription factors necessary for the
expression of the IL-1
gene. Along this line, the recent report
showing that microtubule depolymerization could activate NF
B in HeLa
cells (9) is of particular interest, owing to the importance of this
factor in the control of the IL-1
gene (36).
To gain information on the signal propagation from the microtubules to
the nucleus, we focused our attention on the MAPK congeners that are
known to play a key role in the activation of several transcription
factors, including NFB and NF-IL-6 (37, 38). Several features of
MAPKs substantiate an inter-relationship between the activation of this
kinase family and microtubule remodeling: (i) about 40% of ERK1 and
ERK2 are associated to the microtubules (39); (ii) the best
characterized cellular factors that regulate microtubule dynamics are
MAPs, phosphorylation of which by MAPKs has been proposed to play a
major role in the microtubule growing/shrinking balance (40).
Owing to the important role that was ascribed to SAPK2/P38 activity in
the control of the LPS-induced IL-1 production (11), we first
investigated the possibility that this subfamily of MAPKs was also
involved in the colchicine-induced IL-1 up-regulation. However, we
found that no SB 203580-sensitive activity was recovered in
anti-SAPK2/P38 immunopellets, ruling out a possible involvement of this
type of activity in the mediation of the colchicine effect. In
contrast, ERK1 and ERK2 were activated upon microtubule disruption following two distinct kinetic profiles. ERK2 appears to be activated transiently, while ERK1 was activated in a more sustained fashion. These kinetic profiles perfectly matched the respective translocation pattern of the two MAPK species to the nucleus. We demonstrated that
not only ERK1 and ERK2 were activated, but activators situated upstream, such as MEK-1, Raf-1, and Ras, were also stimulated upon
microtubule depolymerization with a kinetic profile compatible with
that observed for ERK activation. cAMP-dependent protein kinase has been found to be activated upon microtubule disruption (1,
2), and this could lead to directly activate ERK activity as reported
in some systems (41, 42). Our data suggest instead that the activating
effect of colchicine on ERKs does stem from the activation of the
entire MAPK cascade, rather than the mere activation of ERK via
cAMP-dependent protein kinase. We cannot exclude the
possibility that other SAPK congeners such as SAPK1/JNK1, SAPK3, and
SAPK4 (43) are also activated in response to colchicine treatment.
However, when THP1 cells were exposed to PD 98059, a specific inhibitor
of MEK-1 (22), and by way of consequence a blocker of the ERK1/ERK2
pathway, we totally blocked the stimulatory effect of the
vinca-alkaloid on the expression of IL-1 RNA. These data provide the
first evidence that activation of ERK activities is a mandatory
condition for the up-regulation of IL-1
transcription. This agrees
well with the notions that (i) MAPK dramatically up-regulates NF-IL-6
transactivating activity through phosphorylation of its Thr-235 residue
(37) and (ii) NF-IL-6 plays an essential role in the induction of the
human pro-IL-1
gene by interacting with motifs situated on upstream
and proximal regions of the promoter (44).
In an attempt to understand the mechanism by which microtubule
disruption could activate Ras or acts on a step situated upstream, we
investigated a possible involvement of tyrosine kinase activity(ies), which have been shown to act as potent activators of Ras through the
Grb-2/Sos pathway (45, 46). This hypothesis was supported by (i) the
observation that tyrosine kinase inhibitors such as herbimycin or
genistein abolished the colchicine-induced IL-1 transcription; and
(ii) the fact that pervanadate, a potent inhibitor of tyrosine
phosphatases, synergized with the activating effect induced by
suboptimal concentration of colchicine. When THP1 cells were exposed to
colchicine, they exhibited a complex tyrosine phosphorylation pattern
including a set of phosphoproteins migrating in the range of 55-60 kDa
that was reminiscent of tyrosine kinases of the Src type. We could
indeed demonstrate that both c-Src and Hck tyrosine kinase activities
were transiently stimulated with a time course compatible to that
observed for the activation of the MAPK cascade. Activation of c-Src
and Hck was found to be of crucial importance for the mediation of the
colchicine effect since addition of CP 118556, which inhibits Src-like
kinase activities (34), also abrogated the increase in IL-1
transcription induced by microtubule disruption. When we looked to
other members of the Src kinase family expressed in THP1 cells, we
found that exposure to colchicine had no effect on the level of Fyn
activity while that of Lyn was slightly decreased (data not shown),
thus suggesting that, depending on their degree of assembly, tubulin
dimers exert a fine regulation among the Src tyrosine kinase congeners.
Although colchicine treatment does not correspond to a physiological
condition, modulation of microtubule stability does occur under
physiological or pathological conditions. For instance, adherence of
avian marrow macrophages to fibronectin or vitronectin-coated surfaces
that is known to reorganize the microtubule network has been reported to induce the association of c-Src to polymerized tubulin in a tyrosine
kinase independent manner (47). Additionally, we could demonstrate that
pretreatment of THP1 cells with CP 118556 abrogated at the same time
c-Src activity and ERK1 and ERK2 activities, strongly suggesting that,
consecutive to microtubule disruption, c-Src acts as a positive
regulator situated upstream of the ERK activities.
In conclusion, microtubule disruption by vinca-alkaloids appears as a valuable tool to decipher the pathways specifically involved in IL-1 gene regulation in human monocytes, which points to a major role played by the Src-triggered ERK cascade under colchicine treatment. Given that SAPK2 was shown to be essential for the production of the IL-1 under LPS stimulation, our data suggest that, depending on the site inflammatory stimulus, IL-1 production might be controlled by distinct MAPK congeners.
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. S. Kadin for kindly providing CP 118556 and Dr. E. Van Obberghen-Schilling and B. Lantéri for critical reading of the manuscript. We are also grateful to R. Mescatullo and A. Grima for illustration work.
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FOOTNOTES |
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* This work was supported by INSERM, the Ligue Nationale Contre le Cancer Comité Départemental du Var, and the Féderation Nationale des Groupements des Entreprises Françaises dans la Lutte Contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 334-93-37-77-03; Fax: 334-93-81-94-56; E-mail: rossi{at}unice.fr.
1 The abbreviations used are: IL, interleukin; NF, nuclear factor; SAPK, stress-activated protein kinase; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAP, microtubule-associated protein; MAPK, MAP kinase; MEK, MAP kinase kinase; PAGE, polyacrylamide gel electrophoresis; MBP, myelin basic protein.
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REFERENCES |
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