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*VINCRISTINE
The Journal of Immunology, 1999, 163: 5079-5085.
Copyright © 1999 by The American Association of Immunologists

Src-Regulated Extracellular Signal-Related Kinase and Syk-Regulated c-Jun N-Terminal Kinase Pathways Act in Conjunction to Induce IL-1 Synthesis in Response to Microtubule Disruption in HL60 Cells1

Béatrice Cambien, Marie-Ange Millet, Heidy Schmid-Antomarchi, Nicole Brossette, Bernard Rossi2 and Annie Schmid-Alliana

Institut National de la Santé et de la Recherche Scientifique Unite 364, Nice, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A microtubule reorganization is often observed during cellular contacts that are associated to IL-1 production. Here, we show that in HL60 cells, vincristine, a microtubule-disrupting agent that induces a strong production of IL-1, triggers the activation of both extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK-1). While ERK activation is rapid and transient, peaking at 10 min, the JNK1 activation is delayed and more sustained reaching a maximum at 2 h. ERK activation was blocked by CP 118556, indicating it is regulated by a Src-like kinase, while JNK1 was inhibited by piceatannol, revealing an upstream regulation by Syk. Each kind of the nonreceptor tyrosine kinase blockers efficiently inhibits the vincristine-induced IL-1 production and diminishes the level of IL-1 transcripts, indicating that the ERK and JNK pathways act coordinately to elicit the transcription of the IL-1 gene. Furthermore, we found that pertussis toxin, a blocker of Go/Gi proteins, abrogated the vincristine-induced activation of both Src and Syk. Our data support a model where the status of microtubule polymerization influences the activity of Go or Gi proteins that control, in turn, two independent Src/ERK and Syk/JNK1 cascades that are both necessary to sustain IL-1 synthesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-1 is a key mediator in inflammatory and immunological responses (for review see Ref. 1) the primary source of which is the monocyte macrophage lineage. This cytokine is induced by a variety of agents including bacterial endotoxin (LPS), Ag-Ab complexes and a ß-chemotactic factor monocyte chemoattractant protein-1 (2). Regulation of IL-1 synthesis is complex, and the signaling pathways involved in this process are still ill-defined, probably because of the complexity of the signaling induced under the stimulation of natural inflammatory agents.

Microtubules, which represent one major components of the cytoskeleton, are subject to constant remodeling. Depolymerization of microtubules with drugs such as nocodazole and colchicine is shown to activate, at the transcriptional level, several genes including that encoding IL-1ß (3, 4). Among the different cytokines, microtubule-disrupting agents selectively trigger the production of IL-1 in monocytes (4), a situation that simplifies the analysis of the signaling that intervenes specifically in the regulation of this cytokine. A profound microtubule reorganization has been reported to occur in endothelial cells in response to injury (5, 6), in osteoclasts during adherence (7), or during polarization of T cells toward APCs (8). Thus the question arises of the importance of the microtubule remodeling in conveying the signals generated by cell-cell contacts to produce IL-1.

Mitogen-activated protein kinases (MAPKs)3 are divided into two major classes: the extracellular signal-related kinases (ERKs) (for review see Ref. 9) and the stress-activated protein kinases (SAPKs) (9, 10). The ERK pathway is found ubiquitously in eukaryotic organisms, and this cascade can be activated by a variety of receptors, including receptors endowed with tyrosine kinase (RTKs) activity and G protein-coupled receptors (GPCRs). It is well established that the activation of ERKs from RTKs involves a linear cascade including the Src homology 2/3 adapter proteins, guanidine nucleotide exchange factors, p21ras, Raf-1, and MAP/ERK kinase (MEK) (11). However, recent reports indicate that signaling pathways involving phosphatidylinositol 3-kinase or protein kinase C can also phosphorylate MEK and ERKs independently to the p21ras pathway (12, 13, 14). Although activation of the ERK pathway by RTKs is well defined, the mechanism used by heterotrimeric GPCRs to activate this pathway is a matter of intense research (15). Recently, genetic and biochemical evidence has accumulated showing that ligands interacting with GPCR activate different tyrosine kinases that bridge the G proteins to the ERK pathway (16, 17, 18). However, the relative contribution of these kinases to the GPCR signaling leading to MAPK activation is still under investigation. GPCRs have also been demonstrated to stimulate the JNK/SAPK1 pathway (19, 20, 21, 22), the activation of which is under the control of Rac-1 and Cdc42 (19, 23).

In this study, we sought to determine the nature of the MAPK that participate to the propagation of the vincristine-induced signal leading to IL-1 production in human promyelocytic HL60 cells. We present evidence that cytoskeletal reorganization triggers two independent pathways leading respectively to ERK and JNK1 activation, which are both necessary to sustain IL-1 production, whereas JNK2 remains unaffected. We could demonstrate that, while the ERK pathway is under the control of Src, the JNK pathway was independently regulated by Syk. Interestingly, microtubule disruption-mediated activation of both tyrosine kinases was abrogated by pertussis toxin (PTX), suggesting that the state of microtubule polymerization might regulate steps situated upstream of Src and Syk by interfering with the degree of activation Go/Gi proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

HL60, human promyelocytes, were grown in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated FCS (Life Technologies), L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml). FCS was tested for the absence of endotoxin (<0.1 IU/ml; Institute J. Boy, Reims, France). Cells were maintained at 37°C in a humidified 5% CO2 atmosphere.

Measurement of IL-1ß production

HL60 cells (5 x 105 cells/ml) were stimulated for 18 h in 0.5 ml RPMI 1640 medium (48-well plates; Nunc, Naperville, IL) in the presence of effectors. IL-1ß production (cell-associated and secreted forms) was assayed in the cell culture medium by using a specific IL-1ß sandwich ELISA as previously described (24).

RNA isolation and Northern analysis

HL60 cells (106 cells/ml) were stimulated by 1 µM vincristine for 18 h in 5 ml RPMI medium in the presence of effectors. Total cellular RNAs were isolated by guanidinium isothiocyanate-phenol-chloroform extraction method using RNA PLUS kit (Bioprobe, France). Northern analysis was performed as previously described (4).

Products

Vincristine, piceatannol, and PTX were obtained from Sigma (St. Louis, MO). PD 98059, the MEK inhibitor, was purchased from New England Biolabs (Beverly, MA). CP 118556 (also named PP2) was kindly provided by S. Kadin (Pfizer Research, Groton, CT).

Cell stimulation and cell lysis

HL60 cells (7 x 105 cells/ml) were starved 16 h in RPMI 1640 medium and harvested by centrifugation for 5 min at 1000 x g before being resuspended in RPMI 1640 at a concentration of 2 x 107 cells/ml. Cells (107) were treated at 37°C with or without the effectors for the indicated times and lysed at room temperature in a buffer containing 150 mM NaCl, 0.8 mM MgCl2, 5 mM EGTA, 1% Nonidet P-40, 1 mM PMSF, 15 µg/ml leupeptin, 1 µM pepstatine, 1 mM Na3VO4, and 50 mM HEPES at pH 7.5. The crude lysates were centrifuged at 18,000 x g for 10 min at 4°C, and the supernatants were precleared with rabbit nonimmune serum prebound to protein A-Sepharose (Pharmacia-LKB Biotechnologies, Uppsala, Sweden). The precleared lysates were incubated at 4°C for 3 or 16 h with Abs raised against the various transduction proteins previously, or not, bound to protein A-Sepharose. All Abs were used at dilution 1/200.

Immune complex kinase assay

Src kinase activity. Precleared lysates were incubated at 4°C for 4 h with anti c-Src Abs (Santa Cruz Biotechnology, Santa Cruz, CA) followed by the addition of protein A-Sepharose, and then incubated for one additional hour at 4°C. The immunopellets were washed twice with lysis buffer and twice with tyrosine kinase buffer (10 mM MnCl2, 20 mM HEPES, pH 7.5). Samples were then resuspended in 50 µl of tyrosine kinase buffer supplemented with 1 mM DTT and 0.1 mg/ml of acid-denatured enolase, which was used as an exogenous substrate. The kinase assay was started by the addition of 3.75 µM ATP and 20 µCi/ml [{gamma}-32P]ATP (370 MBq/ml; ICN Pharmaceuticals, Costa Mesa, CA). After 15 min at 30°C, the reactions were stopped by the addition of 25 µl of 9x Laemmli sample buffer and boiling for 3 min.

Syk kinase activity. Syk was immunoprecipitated with appropriate polyclonal Abs (Santa Cruz Biotechnology) under the same conditions as described for Src kinase activity. After washing, the immunopellets were resuspended in 50 µl of tyrosine kinase buffer supplemented with 1 mM PMSF and 1 mM p-nitrophenyl phosphate. The autophosphorylation kinase assay was started by addition of 2 µM ATP and 20 µCi/ml [{gamma}-32P]ATP and stopped after 10 min at 30°C by addition of 9x Laemmli sample buffer.

MAPK activities. MAP-related kinases were immunoprecipitated from precleared lysates by incubation at 4°C for 16 h with anti-ERK1, anti-ERK2, anti-JNK1, or anti-JNK2 antisera (Santa Cruz Biotechnology) bound to protein A-Sepharose. Immunopellets were washed twice with lysis buffer, twice with MAPK 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 MAPK buffer containing 30 mM magnesium-acetate in the presence of 0.2 mg/ml of myelin basic protein (MBP; Sigma) or 0.5 mg/ml of GST-ATF2, which were used as exogenous substrates for ERKs or JNKs, respectively. The kinase assay was initiated by the addition of 25 µM ATP and 20 µCi/ml [{gamma}-32P]ATP and stopped, by addition of 9x Laemmli sample buffer, after incubation at 30°C for 30 min for ERKs and 60 min for JNKs, respectively.

Western blotting

For all kinase activities, immune complex reactions were separated on SDS-PAGE (10–15% gel) followed by blotting onto nitrocellulose membrane and autoradiography using hyperfilms (Amersham, Arlington Heights, IL). The respective amount of immunoprecipitated kinases was evaluated by Western blotting. The membranes were incubated overnight at 4°C with polyclonal-specific anti-cSrc, -Syk, -ERK1, -ERK2, -JNK1, or -JNK2 (0.1 µg/ml; Santa Cruz Biotechnology). After three washes with TNN buffer (10 mM Tris/HCl, pH 7.4, 0.15 M NaCl, 1% Nonidet P-40), the primary Abs were detected with HRP-conjugated anti-rabbit (1:10,000) and visualized by enhanced chemiluminescence detection system with autoradiography multipurpose hyperfilms (Amersham).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Microtubule disruption in HL60 cells stimulates ERK and JNK activities following distinct time courses

We and others have shown that microtubule-disrupting drugs are capable of generating a cascade of events that leads to the selective induction of IL-1 synthesis in human monocytes and in THP1 cells (3, 4). Moreover, in these two cellular systems, we could demonstrate that microtubule depolymerization, which was without any effect on SAPK2, can induce by itself the activation of the entire cascade leading to ERK activation (25). To approach the regulation of ERKs by microtubules in promyelocytic HL60 cells, ERK1 and ERK2 were immunoprecipitated from cells exposed to vincristine, and their ability to phosphorylate MBP, a standard substrate for ERKs, was studied in vitro. The data presented in Fig. 1Go, A and B shows that ERK1 and ERK2 activities were stimulated in response to microtubule disruption by vincristine. ERKs activation was transient, peaking at 10 min for ERK1 as well as for ERK2. In parallel studies, JNK1 and JNK2 activities were also assessed in immunopellets using GST-ATF2 as substrate (Fig. 2Go). We observed that microtubule depolymerization, which failed to stimulate JNK2 activity (data not shown) in HL60 cells, activated JNK1 in a time-dependent manner (Fig. 2Go, A and B). Nevertheless, the JNK1 activation kinetic profile was markedly delayed compared with the time course observed for ERKs, being detectable only after 1 h, reaching a maximum by 2 h, which persisted for up to 4 h. Each point of the kinetic for ERK1 and ERK2 (Fig. 1GoB) and for JNK1 (Fig. 2GoB) activities were corrected for small variations in the total amount of the respective kinases present in each immunopellet sample.



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  		FIGURE 1. Microtubule disruption stimulates ERK activities in HL60 cells. A, Time course of vincristine-induced stimulation of ERK activities. Cells, untreated (lane 1) or stimulated for 5 min (lane 2), 10 min (lane 3), 30 min (lane 4), 40 min (lane 5), or 60 min (lane 6) with 1 µM vincristine, were immunoprecipitated with ERK1 or ERK2 Abs. The immune complexes were subjected to in vitro kinase assays using MBP substrate. The 32P-labeled proteins were resolved on 12% SDS-PAGE, blotted onto nitrocellulose membrane, and autoradiographied (immunoprecipitated (IP) ERKs). The same blot was then hybridized with anti-ERK1 or anti-ERK2 Abs (WB ERKs). The autoradiographs relate to a representative experiment among two experiments. B, Densitometric scanning of ERK1 ({triangleup}) and ERK2 ({blacktriangleup}) activities toward MBP. The bands corresponding to MBP phosphorylation were subjected to densitometric scanning and expressed as fold stimulation of the basal level determined in unstimulated HL60 cells. Each value was corrected for small variations in the amounts of ERK1 or ERK2 present in each immunopellet, as assessed by Western blotting analysis. Error bars represent the mean ± SD of two experiments.

 


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  		FIGURE 2. Vincristine induces JNK1 activation in HL60 cells. A, Time course of vincristine-induced stimulation of JNK1 activity. Cells, untreated or stimulated for varying time periods with 1 µM vincristine, were immunoprecipitated with anti JNK1 Abs. The immune complexes were subjected to in vitro kinase assay using GST-ATF2 substrate. The 32P-labeled proteins were resolved on 12% SDS-PAGE, blotted onto nitrocellulose membrane, and autoradiographied (IP JNK1). The same blot was then hybridized with anti-JNK1 Abs (WB JNK1). Autoradiographs relate to the most representative experiment of a series (n = 5). B, Densitometric scanning of JNK1 activity toward GST-ATF2. The results are expressed as fold stimulation of the basal level measured in unstimulated HL60 cells. Each value was corrected for small variations in the amount of JNK1 present in each immunopellet, as estimated by Western blotting with an anti-JNK1 Ab. Error bars represent the mean ± SD of five experiments.

 
In contrast to JNK1, Src-like kinases are involved in the activation of ERKs

We have previously reported in THP1 cells that Src kinases control the stimulation of ERK activities elicited by microtubule depolymerization (25). We confirmed, by the use of CP 118556, a potent inhibitor of the Src kinase family (26), that in the HL60 model, Src-like kinases do activate ERK activities upon microtubule depolymerization (Fig. 3GoA). Moreover, as in THP1 cells (25), activation of Src-like kinases was found to be of crucial importance for the mediation of the vincristine effect because pretreatment of HL60 cells by CP 118556 also abrogated the IL-1ß production (Fig. 3GoB) and the IL-1ß transcription (Fig. 3GoC) that follow microtubule disruption. In the light of these data, we tested whether Src activation was an essential event in vincristine-induced JNK1 stimulation. To this end, HL60 cells were treated with CP 118556 for 2 h before stimulation by vincristine. As shown in Fig. 4Go, A and B, instead of exerting an inhibitory effect, treatment with CP 118556 potentiated the stimulating effect that vincristine exerts on JNK1 activation, although it did not modify the time course of stimulation of this kinase.



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  		FIGURE 3. Activation of Src-like kinases is essential both for the stimulation of ERKs and for the synthesis of IL-1ß by HL60 cells. A, HL60 cells not treated (lanes 1–3) or preincubated 2 h with 30 µM CP 118556 (lanes 4–6) were stimulated for 10 min (lanes 2 and 5) or 30 min (lanes 3 and 6) with 1 µM vincristine. Cell lysates were immunoprecipitated with an anti-ERK1 or anti-ERK2 (n = 3). The activities and the amount of ERKs in immunoprecipitates were evaluated as described in Materials and Methods. B, Effect of CP 118556 on vincristine-induced IL-1ß production by HL60 cells. Cells were stimulated for 18 h by 1 µM vincristine in the presence of various concentrations of CP 118556. Error bars represent the mean ± SD from data of three experiments. C, Northern blot analysis of the effects of CP 118556 on the level of vincristine-induced IL-1ß RNAs in HL60 cells. Cells were untreated (lane 1) or stimulated for 18 h with 1 µM vincristine (lanes 2–5) in the absence (lane 2) or in the presence of 3 µM (lane 3), 10 µM (lane 4), or 30 µM (lane 5) of CP 118556. This panel shows the level of IL-1ß RNA (upper box) and the level of ethidium bromide-labeled ribosomal 18S RNA (lower panel) as a control of the total RNA content.

 


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  		FIGURE 4. Src-like kinases are not involved in the vincristine-induced JNK1 activation. A, HL60 cells not treated (lanes 1–5) or preincubated 2 h with 30 µM of CP 118556 (lanes 6–10) were not exposed (lanes 1 and 6) or exposed 1 h (lanes 2 and 7), 2 h (lanes 3 and 8), 3 h (lanes 4 and 9), and 4 h (lanes 5 and 10) to 1 µM vincristine. At the end of the incubation period, cells were solubilized and JNK1 was immunoprecipitated using specific Abs. The kinase activity of immunoprecipitated JNK1 was assessed using exogenous GST-ATF2 as substrate. The 32P-labeled proteins were resolved on SDS-PAGE, blotted onto nitrocellulose membrane, and autoradiographied (IP JNK1). The same blot was then hybridized with anti JNK1 Abs (WB JNK1). B, Densitometric scanning of vincristine-induced JNK1 activity as expressed as the stimulation factor calculated at each point by dividing the value obtained in the presence of CP 118556 by that observed in the absence of this inhibitor. Each value was corrected for small variations in the amounts of JNK1 present in each immunopellet. Error bars represent the mean ± SD from data of two experiments.

 
Microtubule-disruption activates Syk kinase in HL60 cells

In human peripheral blood monocytes, cell adherence to extracellular matrix via integrins results in the rapid induction of multiple inflammatory mediator genes including several cytokines and in particular IL-1ß (27, 28). It has been reported that stimulation of monocytic cells through integrin cross-linking induces a significant increase in the activity of the Syk kinase (29), a nonreceptor tyrosine kinase (30). These observations prompted us to evaluate whether, in promyelocytic HL60 cells, Syk activation is a prerequisite for microtubule disruption-induced IL-1ß production. Measurement of the Syk kinase activity was achieved by immunoprecipitating the enzyme with specific Abs from unstimulated or vincristine-treated HL60 cell lysates and testing their ability to undergo autophosphorylation. Stimulation of HL60 cells by vincristine induced an increase in the level of Syk autophosphorylation (Fig. 5Go, A and B). This increase did result from an autocatalytic process because it was prevented by piceatannol, a specific inhibitor of the Syk tyrosine kinase family (31). Syk activation reached a plateau value at 15 min that was maintained for up to 60 min. In an attempt to demonstrate that the vincristine-induced activation of Syk was related to the stimulatory effect of the vinca-alcaloid on IL-1ß synthesis, HL60 cells were treated with various concentrations of piceatannol before microtubule disruption. Under these conditions, vincristine-induced IL-1ß production (Fig. 6GoA) was inhibited in a concentration-dependent manner with a half-maximal inhibition of 0.3 µg/ml, in accord with the reported effect of piceatannol on Syk activity as measured in intact cells (31, 32). Blockade of IL-1 production reflected mainly an inhibition of IL-1 transcription as shown in Fig. 6GoB, where the level of IL-1 transcript decreases abruptly at a concentration of 1 µg/ml of piceatannol.



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  		FIGURE 5. Microtubule disruption activates Syk kinase in HL60 cells. A, Cells not treated (lanes 1–6) or preincubated 2 h with 5 µg/ml of piceatannol (lanes 7–12), were not exposed (lanes 1 and 7) or exposed 5 min (lanes 2 and 8), 10 min (lanes 3 and 9), 15 min (lanes 4 and 10), 20 min (lanes 5 and 11), and 60 min (lanes 6 and 12) to 1 µM vincristine. Cell lysates were immunoprecipitated with anti-Syk Abs, and immune complexes were subjected to autophosphorylation activity. The 32P-proteins were resolved on 10% SDS-PAGE followed by blotting and autoradiography (IP Syk). The same blot was then hybridized with anti-Syk Abs (WB Syk) to evaluate the total amount of the kinase in each sample. Autoradiographs relate to a representative experiment among a series (n = 3). B, Densitometric scanning of Syk autophosphorylation. Each value was corrected for small variations in the amounts of Syk present in each immunopellet. Error bars represent the mean ± SD of three experiments.

 


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  		FIGURE 6. Activation of Syk is an essential step for the synthesis of IL-1ß by HL60 cells. A, Effect of piceatannol on vincristine-induced IL-1ß production by HL60 cells. Cells were stimulated for 18 h by 1 µM vincristine in the presence of various concentrations of piceatannol. The production of IL-1ß was assayed in triplicate in the same experiment as described in Material and Methods. Error bars represent the mean ± SD from data of four experiments. B, Northern blot analysis of the effects of piceatannol on the level of vincristine-induced IL-1ß RNAs in HL60 cells. Cells were untreated (lane 1) or stimulated for 18 h with 1 µM vincristine (lanes 2–5) in the absence (lane 2) or presence of 0.01 µg/ml (lane 3), 0.1 µg/ml (lane 4), or 1 µg/ml (lane 5) of piceatannol. This panel shows the level of IL-1ß RNA (upper box) and the level of ribosomal 18S RNA (lower panel) visualized by ethidium bromide as a control of the total RNA content.

 
Syk is required for the JNK but not for the ERK activation elicited by microtubule disruption

Because microtubule depolymerization activated Syk activity, we wished to verify whether this nonreceptor tyrosine kinase was also implicated in the vincristine-induced MAPKs activation. HL60 cells were thus treated with piceatannol before being exposed for 1, 2, 3, and 4 h to vincristine. As shown in Fig. 7GoA, piceatannol abrogated the vincristine-induced JNK activation indicating that Syk controlled this pathway. In contrast, during the entire time span of the vincristine stimulation, piceatannol had no significant effect on the ERK1 or ERK2 activities (Fig. 7GoB). These data demonstrate that ERK and JNK were independently regulated by Src and Syk tyrosine kinases, respectively.



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  		FIGURE 7. Syk is involved in the vincristine-induced activation of JNK1 but not ERKs. A, HL60 cells, not treated (lanes 1–6) or preincubated 2 h (lanes 7–12) with 5 µg/ml of piceatannol, were not exposed (lanes 1 and 7) or exposed 1 h (lanes 2 and 8), 2 h (lanes 3 and 9), 3 h (lanes 4 and 10), 4 h (lanes 5 and 11), or 5 h (lanes 6 and 12) to 1 µM vincristine. Cell lysates were immunoprecipitated with anti JNK1 Abs. The activity and the amount of JNK1 was evaluated as described in Materials and Methods (n = 3). B, HL60 cells, not treated (lanes 1–6) or preincubated 2 h (lanes 7–12) with 5 µg/ml of piceatannol, were not exposed (lanes 1 and 7) or exposed 5 min (lanes 2 and 8), 10 min (lanes 3 and 9), 30 min (lanes 4 and 10), 40 min (lanes 5 and 11), or 60 min (lanes 6 and 12) to 1 µM vincristine. Cell lysates were immunoprecipitated with anti-ERK1 or anti-ERK2 Abs. The activities and the amount of ERKs were evaluated as described in Materials and Methods (n = 3).

 
Heterotrimeric G proteins control the vincristine-induced activation of both Src and Syk kinases, which regulate ERK and JNK kinases, respectively

Previous studies have shown that heterotrimeric G proteins regulate both ERK and JNK pathways in many cell types. Furthermore, G proteins have been shown to mediate MAPKs activation through PTX-sensitive and -insensitive pathways (for review see Refs. 33 and 34).

To precise whether G proteins were implicated in the differential control of ERK and JNK, HL60 cells were first treated, for 4 h before the stimulation by vincristine, with PTX, an inhibitor of {alpha}i and {alpha}o G proteins. We verified that this treatment was without any effect on cell viability as assessed by measuring the activity of lactate dehydrogenase in the supernatant (data not shown). The MAPKs were immunoprecipitated with specific Abs from unstimulated or vincristine-treated HL60 cell lysates and then tested for their ability to phosphorylate in vitro either MBP or GST-ATF2. The data presented in Fig. 8Go shows that treatment with PTX dramatically diminished the vincristine-induced activation of ERK1 and ERK2 (Fig. 8GoA) as well as JNK1 (Fig. 8GoB). These findings support the conclusion that in HL60 at least one PTX-sensitive Go/Gi protein controlled the ERKs and JNK1 activation in response to microtubule disruption.



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  		FIGURE 8. PTX inhibits vincristine-induced ERK and JNK activation. HL60 cells, not treated (no toxin) or preincubated 4 h with 250 ng/ml PTX were stimulated for different times with 1 µM vincristine and lysed. Cell lysates were immunoprecipitated with anti-ERK1 or anti-ERK2 Abs in A (n = 3) and with anti-JNK1 Abs in B (n = 3). The activities and the amount of ERKs and JNK1 in immunoprecipitates were evaluated as described in Materials and Methods.

 
Given that Src and Syk kinases were shown to be situated upstream of the vincristine-induced ERK and JNK pathways, respectively, we investigated the effects of PTX treatment on these two tyrosine kinase activities. We observed that stimulation of both Syk (Fig. 9GoA) and c-Src (Fig. 9GoB) by vincristine was dramatically inhibited in the presence of PTX. These observations highlight the importance of the status of the microtubule polymerization in the regulation of heterotrimeric G proteins that control in turn the level of Src and Syk activities.



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  		FIGURE 9. Pretreatment of HL60 cells by PTX abrogates vincristine-induced c-Src and Syk activities. HL60 cells, not treated (no toxin) or preincubated 4 h with 250 ng/ml PTX, were stimulated for varying time intervals with 1 µM vincristine and lysed. A, Cell lysates were immunoprecipitated with anti Syk Abs (n = 2). The activities and the amount of Syk in immunoprecipitates were evaluated as described in Materials and Methods. B, Cell lysates were immunoprecipitated with anti c-Src Abs. Src activity toward enolase was evaluated in immunoprecipitates, and the amount of c-Src was estimated from lysates as described in Materials and Methods (n = 3). Curiously, the anti-c-Src Ab used in this experiment reacted with three distinct bands, but we verified with another batch of Ab that this does not change the evaluation of the amount of the kinase.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Monocytes are the principal source of IL-1, which is acknowledged to play a major role in the inflammatory process. However, to date the signaling related to the regulation of the IL-1 genes (IL-1{alpha} and IL-1ß) remains elusive, probably because potent natural inducers of IL-1 like LPS also induce the concomitant expression of various inflammatory cytokines such as TNF-{alpha}, IFN-{gamma}, and IL-6. In this situation, it is difficult to ascribe a defined signaling pathway to the regulation of a given cytokine. We and others have described previously that microtubule-disrupting agents can induce the selective production of IL-1{alpha} and IL-1ß in human monocytes at the exclusion of other inflammatory cytokines (4). This represents an ideal model for studying the signaling pathways that specifically intervene in the regulation of the IL-1 genes.

Recently, we have reported that microtubule disruption produces the activation of Src kinases, which in turn controls the activation of the Ras/ERK cascade, resulting ultimately in the augmentation of IL-1ß synthesis (4). In an attempt to further characterize the signaling pathways intervening specifically in the up-regulation of IL-1, we have investigated the possible involvement of other MAPK congeners. In fact, we found that in addition to the ERK activation, microtubule disruption also produced a dramatic increase in the JNK1 activity as assessed by the capacity of the immunoprecipitated JNK1 to phosphorylate recombinant GST-ATF2. The kinetic profiles of the two types of MAPK were very different with a swift and transient activation for ERK, while JNK1 presented a slower and more sustained activation time course. Under the same conditions JNK2 remained unaffected (data not shown). Moreover, at variance to what we observed with ERKs, microtubule disruption-mediated JNK1 activation not only was resistant to inhibitors of Src-like kinases, such as CP 118556 (26), but also exhibited an increased activity in the presence of this inhibitor, suggesting a repressive control of Src-like kinases on the JNK1 pathway.

Stimulation of monocytic cells through integrins has been reported to significantly increase the activity of the Syk kinase (29). Recently, Syk has been reported to cooperate with Rac to activate the JNK pathway upon CD28-mediated T cell stimulation (35). We verified, in our system, that Syk activity was also increased in response to vincristine treatment, as measured by immunoprecipitating the enzyme from lysates of cells exposed to the microtubule disrupting drug. Furthermore, we could demonstrate that Syk acts upstream of the JNK pathway because piceatannol, a specific inhibitor of the Syk tyrosine kinase family (31), abrogated the vincristine-induced JNK activation. The fact that piceatannol also dramatically diminished the vincristine-induced up-regulation of IL-1 transcripts in the same concentration range highly suggests that the Syk/JNK pathway is an absolute prerequisite for IL-1 transcription. Blockade of the Syk kinase by piceatannol had no effect on ERK activity, ruling out the possibility that Syk mirrored, on the Ras/ERK pathway, the inhibitory effect that c-Src exerted on the JNK pathway.

Inasmuch as activation of the ERK pathway was also shown to be indispensable for IL-1 production (25), our data lend support to a model where the positive control of IL-1 synthesis necessitates the coordinated activation of the ERK and JNK pathways. The fact, that the two activities are not activated at the same time suggest the existence of a sequential process.

Remodeling of microtubules has been observed under various situations, implying adherence of hemopoietic cells (7, 8), but the mechanisms through which remodeling of microtubules can activate nonreceptor tyrosine kinases remains to be elucidated. Indeed, c-Src associates to microtubules upon osteoclast adherence (36), Syk interacts with {alpha}-tubulin in B cells once activated by cross-linking the B cell Ag receptor (37), and ZAP-70, a member of the Syk family, is constitutively associated to tubulin in T cells (38). Furthermore, growing evidence has accumulated in the recent years showing that ligands that interact with GPCRs induce the activation of nonreceptor tyrosine kinases. In avian B lymphoma cells, Syk has been shown to be activated via a muscarinic acetylcholine receptor coupled either to Gq (m1 mAChR) or to Gi (m2 mAChR), while in the same system Src-related kinases are activated through a single Gq-regulated pathway (17).

We thus addressed the question whether the activation of Syk and c-Src, in our model, was also under the dependence of trimeric G proteins. To this end, PTX that blocks {alpha}o and {alpha}i G proteins was used. Surprisingly, this toxin dramatically inhibited both the microtubule disruption-mediated ERK and JNK activation, as well as c-Src and Syk tyrosine kinases. These data indicate that 1) the microtubule architecture exerts a potent regulation on the Syk and Src tyrosine kinases and 2) confirm the conclusions obtained by pharmacological means that Syk and Src control JNK and ERK pathways, respectively.

It is noteworthy that tubulin, which also belongs to the GTP-binding protein family, has been proven to directly associate to G{alpha}s and G{alpha}i, thus regulating their activating and inhibiting activity, respectively, on adenylate cyclase activity (39). More precisely, the {alpha} subunit of Gi1 has been reported to bind weakly with assembled microtubules, whereas it interacts strongly with tubulin dimers at a domain that corresponds to the zone of interaction with the other tubulin dimers in polymerized microtubules (40). We thus hypothesize that tubulin, which interacts on the one hand with non receptor tyrosine kinases and on the other hand with trimeric G proteins, might be the link that, under its unpolymerized form, bridges Gi proteins to c-Src and Syk, resulting in the stimulation of their respective enzymatic activity. Unraveling of the precise mechanisms whereby G-proteins activate tyrosine kinase activities is a major challenge for our future studies.

Taken collectively, our data point to the necessity of a concerted activation of the ERK and the JNK1 pathways to induce the transcription of the IL-1ß gene. These two pathways being controlled by distinct tyrosine kinases, c-Src and Syk, respectively. Furthermore, we provide evidence that the degree of polymerization of the microtubule network is of primary importance for the status of activation of Go/Gi proteins, which act upstream of these kinases and, by the way of consequence, appear of crucial importance for the control of the IL-1 production. This study shed some light on the mechanisms through which IL-1-producing cells react to their environment in term of cytoskeleton remodeling to produce this potent proinflammatory cytokine. In this regard, it is worthy to note that LPS, a potent inducer of IL-1, binds to microtubules via MAP-2 (41) and that an important microtubule reorganization has been reported in a variety of cellular interactions (5, 6, 7, 8) that are susceptible to trigger IL-1 synthesis.


   Footnotes
 
1 This work was supported by the Institut National de la Santé et de la Recherche Médicale and La Ligue Contre le Cancer Comité Départemental du Var. Back

2 Address correspondence and reprint requests to Dr. Bernard Rossi, Institut National de la Santé et de la Recherche Scientifique Unite 364, Faculté de Médecine de Nice, Avenue de Valombrose, 06107 Nice Cedex 02, France. E-mail address: Back

3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-related kinase; SAPK, stress-activated protein kinase; RTK, receptor endowed with tyrosine kinase; GPCR, G protein-coupled receptor; MEK, MAP/ERK kinase; MBP, myelin basic protein; PTX, pertussis toxin. Back

Received for publication February 22, 1999. Accepted for publication August 24, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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