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Volume 270, Number 3, Issue of January 20, 1995 pp. 1261-1268
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Two Distinct Signalling Pathways Are Involved in the Control of the Biphasic junB Transcription Induced by Interleukin-6 in the B Cell Hybridoma 7TD1 (*)

(Received for publication, October 11, 1994)

Roger Rezzonico ,  Gilles Ponzio (§), ,  Agnès Loubat ,  Dominique Lallemand (¶), ,  Amanda Proudfoot (**), ,  Bernard Rossi

From the From INSERM U364, Faculté de Médecine, Avenue de Vallombrose, 06107 Nice Cedex 02, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have measured the level of junB mRNA in the B hybridoma cell line 7TD1, under interleukin-6 (IL-6) stimulation. IL-6 increases junB mRNA in a biphasic fashion. The first early-induced peak was transient and likely corresponds to the well documented typical junB mRNA, stimulated in response to numerous growth factors, including IL-6. At variance, the second peak which has never been reported previously, lasted several hours. As a consequence of its effect on junB mRNA, IL-6 stimulated, in a biphasic fashion, the nuclear accumulation of the JunB protein. In this study, we demonstrated that IL-6 regulation occurred exclusively at the transcriptional level and that the bimodal increase of junB mRNA and JunB protein can be accounted for by a biphasic stimulation of junB transcription.

Furthermore, our data point to two major differences between the mechanism of control of the early and the late IL-6-induced junB transcription waves.

First, cycloheximide strongly potentiated the transcription of the second wave, whereas it failed to affect the early-induced burst. Second, tyrphostin, a tyrosine kinase inhibitor, impaired the expression of the first but not the second junB mRNA peak. Conversely, genistein, another tyrosine kinase inhibitor, totally abolished the expression of the second peak of junB mRNA whereas it did not affect the expression of the first peak.

Altogether these data indicate that, in 7TD1 cells, IL-6 controls junB transcription in a biphasic fashion by means of two separate transduction pathways.

INTRODUCTION

Interleukin-6 (IL-6) (^1)is a cytokine which plays an important role in a wide range of biological activities including B cell differentiation, acute phase response to injury and inflammation(1, 2, 3, 4) , T cell activation, and neuronal cell and macrophage differentiation(5, 6, 7, 8) . It has also been described to promote the growth of some murine hybridoma and plasmocytoma(9, 10, 11) , as well as human myeloma(12) . The signal transduction pathways involved in the mediation of these various effects remain to be elucidated. The IL-6 receptor is composed of an 80-kDa ligand-binding subunit associated with a 130-kDa (gp130) signal-transducing moiety (13, 14) . gp130 contains a large intracytoplasmic domain lacking homology with any known protein kinases or other proteins carrying a catalytic activity. Upon binding, the ligand mediates the dimerization of gp130 and the activation of one tyrosine kinase belonging to the Janus kinase family (Jak1, Jak2, or Tyk2)(15, 16, 17) .

In response to IL-6, several transcription factors are activated (18, 19, 20, 21, 22, 23, 24, 25) . Along this line, factors involved in the regulation of acute phase protein genes have been investigated extensively. The transcription factor referred to as nuclear factor NF-IL-6 (C/EBPbeta) was initially identified as a factor that binds to the AGATTGCACAATCT consensus sequence encompassed within the IL-6 promoter(26) . This factor was involved in the induction of several class 1 acute phase protein genes (alpha(1)-acid glycoprotein, angiotensinogen . . . ) by IL-6(23) . NF-IL-6 is ubiquitous, inducible at the transcription level, and the resulting protein is activated by phosphorylation on threonine residues by microtubule associated protein kinase(s)(27) . Besides NF-IL-6, IL-6 also stimulates the activity of two other factors involved in the transcription of the class 2 acute phase protein genes (alpha(2)-macroglobulin, alpha(1)-antichymotrypsin . . . ). They have been identified as IL-6 response element binding protein (18, 19) and acute phase response factor/stat-3(20, 25, 28) . IL-6 response element binding protein is produced in an activated state several hours after the addition of IL-6, by a mechanism involving protein neosynthesis(19) . In contrast, the activation of acute phase response factor/stat-3 does not require protein synthesis, since this factor is directly phosphorylated by the Jak family protein tyrosine kinase and translocated in the nucleus within minutes following IL-6 stimulation (16) .

Several reports have also demonstrated that IL-6 triggers the tyrosine phosphorylation of a 160-kDa protein that controls in turn the early activation of junB mRNA(29, 30) , one possible component of the transcription factors AP-1 (31) or NFAT(32) . Under IL-6 stimulation, the transcription of early junB is controlled by another transcriptional factor, that interacts with an IL-6-specific cis-regulating element (JRE-IL-6) located at position -149 to -124 of junB promoter(33) . This factor is activated by a H7-sensitive pathway that does not involved protein kinase C, protein kinase A, or microtubule associated protein kinases(33) , suggesting that this IL-6 signaling pathway is different from those evoked for acute phase response factor/stat-3 or NF-IL-6. In this respect, the study of the regulation of junB provides an interesting approach for investigating novel IL-6-dependent pathways.

In the present report we demonstrated that besides its ability to induce early junB, IL-6 can also stimulate a second delayed and sustained wave of expression of junB mRNA. This biphasic increase corresponds to two transcriptional bursts, resulting subsequently in a biphasic increase of the junB-encoded protein (JunB). We also provided evidence that the two waves of junB are regulated by two separated pathways that are controlled by distinct tyrosine kinases.

MATERIALS AND METHODS

Reagents

Recombinant human interleukin-6 (IL-6), purified as described elsewhere(34) , was generously provided by Glaxo Institute for Molecular Biology (Geneva, Switzerland). Culture medium was from Life Technologies, Inc. (Cergy Pontoise, France). Actinomycin D, cycloheximide, genistein, tyrphostin 25 (RG50875), fetal calf serum, and human thrombin were purchased from Sigma. Enhanced chemiluminescence (ECL) Western blotting kit reagent and the radioisotopes [alpha-P]dCTP (3000 Ci/mmol) and [alpha-P]UTP (800 Ci/mmol) were from Amersham (Les Ulis, France). Rabbit affinity-purified polyclonal anti-JunB antibodies (JunB 2.2) used in Western blotting experiments were kind gifts from Dr. M. Yaniv (Institut Pasteur, URA 1644, Paris, France). Peroxidase-conjugated goat anti-rabbit antibodies were from Dako (Denmark). QuickHyb hybridization solution and pBluescript SK/phagemid were purchased from Stratagene. PGEX-4T/2 vector, isopropyl-1-thio-beta-D-galactopyranoside, and glutathione-Sepharose 4B were from Pharmacia/LKB (St-Quentin Yvelines, France).

Cell Culture

7TD1 cells (2.5 to 3.5 times 10^5/ml) were maintained at 37 °C, in 8% CO(2) atmosphere, in Dulbecco's modified Eagle's medium culture medium supplemented with 10% (v/v) fetal calf serum and 100 units/ml IL-6. Within kinetics experiments, cells were first synchronized in G1 phase by depriving them of IL-6 for 16-24 h, then restimulated by the cytokine for indicated times. In experiments using tyrosine kinase inhibitors or transcription and protein synthesis blockers, cell viability was determined in parallel by trypan blue staining.

RNA Extraction and Northern Blot Analysis

Total cellular RNA was prepared by denaturation in guanidinium thiocyanate followed by pelleting through a cesium chloride cushion(35) . For Northern blot analysis, 15-20 µg of total RNAs were loaded on a 1% agarose gel in MOPS buffer, containing 0.7% formaldehyde and transferred onto nylon Hybond N membrane (Amersham). Probe hybridizations (10^6 cpm/ml) were carried out overnight at 65 °C in a QuickHyb solution (Stratagene), filters were washed in 0.1 times SSC, 0.1 SDS at 65 °C for 1 h and finally exposed to Amersham Hyperfilms-MP at -80 °C. Northern blot experiments were quantified by densitometric scanning using a computerized microscopic image processor Biocom 500 (Biocom, Les Ulis, France) comprising a PC/AT-compatible microcomputer, a real time imaging processor, a control monitor, a color high definition monitor, and a Panasonic WV-CD50 camera.

cDNA Probes

The cDNA probe for junB mRNAs was a 0.7-kilobase EcoRI/AccI fragment excised from Rous sarcoma virus-junB, corresponding to the sequence of the clone 465, published by Ryder(36) . The murine c-myc cDNA was a generous gift from Dr. Dani (Nice, France).

Nuclei Isolation and Run-on Transcription Assay

Nuclei were isolated from 7TD1 cells (2-4 times 10^7) and run-on transcription assay performed according to the method described by Doglio et al.(37) . Nylon filters (Hybond N, Amersham) spotted with 5 µg of pBluescript SK containing junB or c-myc cDNA were hybridized for 48 h at 65 °C with 10^7 cpm of biosynthetically P-labeled nuclear RNAs, then washed four times at 65 °C for 2 h with 2 times SSC, 0.1% SDS. Filters were dried and exposed to Amersham Hyperfilms MP at -80 °C.

Preparation of Nuclear Extracts and Immunoblot Analysis

Nuclear extracts were prepared using a method derived from Dignam et al.(38) . Briefly, nuclei (4-5 10^7) were isolated as described above and lysed by the addition of nuclear extraction buffer (20 mM Hepes, pH 7.9, 25% glycerol, 1.5 mM MgCl(2), 0.25 mM EDTA, 0.37 M NaCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 5 µg/ml pepstatin). The suspension was gently rocked for 30 min then centrifuged for 30 min at 25,000 rpm. The supernatants were collected and precipitated overnight by addition of 300 mg/ml ammonium sulfate. Proteins were pelleted by centrifugation for 20 min at 25,000 rpm, resuspended in 100 µl of a buffer containing: 20 mM Hepes, pH 7.9, 60 mM KCl, 20% glycerol, 0.25 mM EDTA, 0.125 mM EGTA, supplemented with protease inhibitors and dialyzed twice for 3 h at 4 °C against the same buffer.

Nuclear protein samples (100 µg) were separated by SDS-polyacrylamide gel electrophoresis, transferred to an Immobilon membrane (Millipore), and incubated first with a specific anti-JunB (JunB 2.2) polyclonal antibody (1:750 dilution) and then with a peroxidase-conjugated goat anti-rabbit antibody (1:2,000 dilution). The transferred proteins were finally detected using an enhanced chemiluminescence detection system (Amersham) according to the manufacturer's procedures.

Expression and Purification of a Bacterially Synthesized Glutathione S-Transferase-JunB Protein and Immunoblotting

This protein was derived from a pGEX-4T vector (Pharmacia) into which the 1.4-kilobase junB cDNA, encoding for the full-length protein, was cloned into SmaI/XhoI sites. Isopropyl-1-thio-beta-D-galactopyranoside (1 mM for 6 h) treatment of bacteria harboring this plasmid grown at 28 °C, induced the expression of soluble 66-67-kDa protein comprising glutathione S-transferase (26-28 kDa) fused with the 39-kDa JunB, respectively. Following lysis by sonication, solubilization with 1% Triton X-100 and centrifugation to remove debris, the protein was absorbed on ice to glutathione-Sepharose (Pharmacia Biotech). The beads were then washed extensively in phosphate-buffered saline containing 0.1% Triton X-100, 0.5 M NaCl. The 39-kDa JunB protein was finally eluted from the column by cleavage with 1 µg/ml human thrombin for 1 h at 25 °C. The protein (50 ng) was subjected to SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to an Immobilon membrane. Immunoblotting was performed as described above with an anti-JunB (JunB 2.2) antibody.

RESULTS

JunB mRNA Is Stimulated in a Biphasic Fashion in Response to IL-6 on 7TD1 Cells

The jun family members junB and c-jun are usually transiently stimulated in the early response to numerous stimuli including IL-6(29, 30, 39) . In recent reports, authors have demonstrated that, besides their early effect on c-jun mRNA, effectors like thrombin or fetal calf serum are also able to elicit a second phase of c-jun mRNA induction(40, 41) .

In an attempt to verify whether IL-6 was also able to stimulate a late induction of junB mRNA in 7TD1 cells, we measured the level of junB mRNA within the 10 h following the addition of IL-6. Northern blot analysis carried out on total RNAs, as described in Fig. 1, panel B, revealed that IL-6 strongly augmented the cellular level of junB mRNA following a bimodal fashion. The first peak of induction reached a maximal value after 1 h and then rapidly declined after 2 h of stimulation in the presence of IL-6. The second phase which was more sustained occurred between 5 and 9 h after stimulation. The stimulation factors estimated by scanning the bands corresponding to junB mRNA were, respectively, around 13-fold for the first peak and 4- to 5-fold for the second peak (Fig. 1, panel A).

Figure 1: Time course of induction by interleukin-6 of junB and c-myc mRNAs. Growing 7TD1 cells were synchronized by IL-6 deprivation and restimulated with the cytokine for the indicated times. Total RNAs were prepared, loaded on agarose gels, blotted onto nylon membrane, and hybridized as described under ``Materials and Methods'' with cDNA probes encoding for, respectively, junB and c-myc. Panel A shows the densitometric scanning of Northern blot experiments of IL-6-induced junB mRNA. Panel B shows the level of junB (upper part) and c-myc (lower part) mRNA estimated by Northern blot analysis, as well as the level of ribosomal 18 S RNA visualized by ethidium bromide as a control of the total RNA level. Autoradiograms presented were obtained after 1 day of exposure.


This biphasic increase was selective since IL-6 did not stimulate or modify the constitutive level of c-myc in the same interval of time (Fig. 1, panel B)

The Biphasic Stimulation of junB mRNA Is Associated with a Biphasic Increase in JunB Protein

To determine whether the biphasic regulation of junB mRNA resulted in a biphasic accumulation of JunB protein, we have assessed, by Western blotting analysis, the JunB protein level in nuclear extracts from 7TD1 cells stimulated with IL-6 (100 units/ml) for 0.5, 1, 2, 4, and 6 h. Results are shown in Fig. 2, panel A. In IL-6-deprived cells, no immunoreactive JunB protein could be detected. The level of protein increased markedly when IL-6 was added, raised a maximal level after 1 h, before declining to a basal level at 4 h. Then the protein level increased again by 6 h, suggesting that the biphasic stimulation of junB mRNA was accompanied by the increase of the nuclear JunB protein.

Figure 2: Kinetics of induction by interleukin-6 of nuclear JunB protein. IL-6-deprived 7TD1 cells were stimulated with IL-6 for the indicated times. Nuclear proteins (100 µg) were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane as described under ``Materials and Methods.'' Immunoblots were then probed either with a specific anti-JunB antibody or a nonrelevant serum (N.I.S.) and developed by enhanced chemiluminescence (ECL) revelation. Panel A shows the biphasic induction by IL-6 of the nuclear JunB protein. Panel B shows the comparative migration pattern of: the nuclear JunB protein of 7TD1 cells, nonstimulated (lane 2) or stimulated (lane 3) for 1 h with IL-6 versus a nonphosphorylated bacterially expressed JunB fusion protein (lane 4). Lane 1 shows nuclear proteins of 7TD1 cells stimulated with IL-6, probed with a nonrelevant serum.


It is currently accepted that the phosphorylation of c-Jun is responsible for its electrophoretic retardation from 39 kDa to 46 kDa (42) . In an attempt to define the phosphorylated status of the nuclear IL-6-translocated JunB protein, we have compared the migration pattern of the protein present in 7TD1 nuclear extracts with that of a nonphosphorylated bacterially expressed fusion protein. Lanes 2 and 3, Panel B represented the stimulation by IL-6 of the nuclear JunB protein. As indicated, JunB migrated as an unique band with an apparent molecular mass of 46 kDa slightly higher than the bacterially expressed protein (39-40 kDa) (lane 3 versus lane 4) suggesting, by analogy with results observed on c-Jun, that IL-6 might stimulate the nuclear translocation of a phosphorylated form of JunB.

IL-6 Stimulates junB Transcription in a Biphasic Manner

To gain information on the way IL-6 elicits the increase of the two junB mRNA peaks, run-on experiments were performed to first determine whether IL-6 controlled the transcriptional rate of this gene. Nascent nuclear RNA chains, biosynthetically labeled with [alpha-P]UTP, were isolated from 7TD1 cells stimulated for 0 to 10 h with IL-6 and used to hybridize nitrocellulose filters previously spotted with plasmids containing either c-myc or junB inserts. Results concerning junB transcription are shown in Fig. 3, panel B. In accordance with previous results (30) , a very low but detectable constitutive level of junB transcription was measurable in 7TD1 cells deprived of IL-6 for 24 h. After the addition of IL-6, junB transcripts increased abruptly to reach a maximal value at 30-60 min, before declining to the basal value by 2-3 h. Interestingly, the transcription resumed after 5 h and peaked at 6 h, before declining again by 8-9 h. The stimulatory factors, as determined by scanning the spots corresponding to the neosynthesized junB transcripts, were, respectively, 8-fold for the early peak (maximum at 30 min) and 4-fold for the second peak (maximum at 6 h) (Fig. 3, panel A). The biphasic time course of the burst in the transcription rate is an original feature that probably accounts for the immediate and delayed increase in junB mRNA levels. In contrast with junB transcription, c-myc gene was constitutively transcribed in noninduced 7TD1 cells and was not further stimulated in the presence of IL-6 (Fig. 3, panel C). Taken together, these results suggest that the biphasic transcription burst, described here, is not a general IL-6-mediated phenomena since it does not concern other genes like c-myc.

Figure 3: Transcriptional induction of junB and c-myc genes in isolated nuclei of 7TD1 cells exposed to IL-6. Quiescent 7TD1 cells were exposed to IL-6 (100 units/ml) for the indicated times. Nuclei were isolated, and [alpha-P]UTP was incorporated into nascent RNA chains as described under ``Materials and Methods.'' Labeled RNA (10^7 cpm) was then hybridized for 48 h to 5 µg of junB (panel B) or c-myc cDNA (panel C) immobilized on nitrocellulose filters. After washing, filters were exposed to x-ray film for 1 day. Panel A represents the densitometric scanning of the autoradiograms hybridized with junB (box) and c-myc () cDNAs, respectively.


IL-6 Does Not Modify the Stability of junB mRNA

In order to further examine the effect of IL-6 on the stability of the two waves of junB mRNA, 7TD1 cells were treated with IL-6 (100 units/ml = 500 pg/ml) for 1 or 6 h, then exposed to actinomycin D (5 µg/ml). The decay of RNA was followed afterward in the presence of IL-6 or after withdrawal of the cytokine. Northern blot analyses are shown in Fig. 4, panels B, C, D, and E (early peak), and Fig. 5, panels B, C, D, and E (late peak). The corresponding densitometric RNA level quantifications are presented in panel A of Fig. 4and Fig. 5. Regardless of the presence of the cytokine or actinomycin D, the first burst of transcription was shut off after 60-90 min, and junB mRNA decayed with the same half-life (about 40-50 min). These data demonstrate that IL-6 exclusively controlled the transcription step without any modification of junB mRNA stability.

Figure 4: Effect of IL-6 on the stability of the early junB mRNA peak. Quiescent 7TD1 cells were stimulated with IL-6 for 1 h, and the level of junB mRNA was measured during the next 3 h, by Northern blot analysis, either in the presence of IL-6 (panel B) or after the transcription was blocked by: IL-6 withdrawal (panel C), actinomycin D addition (panel D), or both IL-6 removal and addition of actinomycin D (panel E). On panel A are represented the densitometric scanning of the Northern blot analysis presented in panels B, C, D, and E, referred, respectively, as box, , circle, and bullet.


Figure 5: Effect of IL-6 on the stability of the late-induced junB mRNA wave. This experiment has been carried out in the same conditions as in Fig. 4, except that cells were stimulated with IL-6 for 6 h instead of 1 h. On panel B is depicted the level of junB mRNA, determined by Northern blot analysis, between 6 and 9 h in the presence of IL-6. Panels C, D, and E show the level of IL-6-induced junB mRNA after the transcription was blocked by: IL-6 withdrawal (panel C), actinomycin D addition (5 µg/ml) (panel D), or both (panel E). Panel A shows densitometric scanning of Northern blots shown in panels B, C, D, and E. The symbols box, , circle, and bullet correspond, respectively, to panels B, C, D, and E.


When the transcription was blocked by actinomycin D, 6 h after the addition of IL-6, junB mRNA decayed with the same time course (half-life about 40 min), whatever the presence or absence of IL-6 in the medium (Fig. 5, panels D and E), suggesting that IL-6 did not control the stability of the second peak of junB mRNA.

Concerning the second wave of junB transcription, it is interesting to notice that as soon as IL-6 was removed junB mRNA decayed rapidly (Fig. 5, panel C) indicating that IL-6 withdrawal led to a rapid arrest of junB transcription. Thus, at variance with the first peak of junB RNA, IL-6 was permanently required to sustain junB transcription during the second wave.

Cycloheximide Potentiated the Effect of IL-6 on the Second but Not on the First junB mRNA Peak

Studies were carried out to investigate whether inhibition of protein synthesis might influence the early and/or late junB transcription and mRNA stability.

Quiescent cells were stimulated with IL-6 for 1 or 6 h, in the presence or absence of cycloheximide (10 µg/ml), and then treated with or without actinomycin D (5 µg/ml) after which the junB transcription and mRNA level were followed for 4 h.

The results demonstrated that the level of the early-induced junB mRNA was identical in cells treated with or without cycloheximide (not shown). As shown in Fig. 6, panel B, when the transcription was blocked by actinomycin D, cycloheximide did not modify the time course of junB mRNA decay (about 40 min as estimated by quantitating the RNA level by densitometric scanning of the autoradiogram, panel A) suggesting that protein synthesis is neither required for the synthesis nor for the degradation phases of this rapid junB mRNA wave. In a parallel run-on experiment, we also demonstrated that cycloheximide did not modify early IL-6-induced junB transcription (data not shown). Our observation is in agreement with previous results from Nakajima and Wall (30) on the MH60 BSF-2 cell line. At variance, it differs from the results obtained by Lord et al.(29) on the myeloid cell line M1. On this model, these authors demonstrated that cycloheximide stabilized junB mRNA by decreasing its degradation.

Figure 6: Effect of cycloheximide on the early-induced junB mRNA peak. Quiescent 7TD1 cells were preincubated for 1 h with cycloheximide (10 µg/ml), stimulated with IL-6 for 1 h, then incubated in the absence (box) or in the presence () of actinomycin D (5 µg/ml). At the indicated times, total RNAs were extracted and the level of junB mRNA was analyzed by Northern blot as described under ``Materials and Methods.'' The nitrocelluloses were exposed to x-ray film for 24 h (panel B). Panel A shows densitometric scanning of Northern blot analysis shown in panel B.


The situation was different when we looked to the effect of cycloheximide on the induction of the second junB mRNA wave. Indeed, in the presence of cycloheximide, the level of the late-induced junB mRNA measured after 6 h of IL-6 was 15-20-fold higher than that measured without protein synthesis inhibitor (Fig. 7, panel A). To approach the mechanism of this superinduction, we measured IL-6-stimulated junB transcription by run-on experiments, after exposure for 6 and 8 h of the cells to IL-6, in the absence or in the presence of 10 µg/ml cycloheximide. Clearly, cycloheximide potentiated the IL-6-stimulated junB transcription rate to an extent that matched mRNA accumulation (Fig. 7, panel B).

Figure 7: Effect of cycloheximide on the late IL-6-induced junB mRNA wave. The figure represents the levels of the second peaks of: junB mRNA (densitometric scanning of an overnight exposed autoradiogram) (panel A) and junB transcription (autoradiogram exposed for 24 h at -80 °C) (panel B) stimulated by IL-6, in the presence or absence of cycloheximide (10 µg/ml). On panel C is shown the effect of cycloheximide on the stability of the late-induced junB mRNA wave. In each experiment, 7TD1 cells were first stimulated with IL-6 (100 units/ml). After 3 h, cycloheximide was added and the incubation was pursued for an additional 3 h, then the levels of junB transcription and mRNA were estimated by run-on experiment and Northern blot analysis as described under ``Materials and Methods.'' To investigate the effect of cycloheximide on junB mRNA stability (panel C), cells were stimulated, by IL-6 in the presence of cycloheximide, according to the procedure described above, then actinomycin was added () or not (box), and the level of junB mRNA was measured at the indicated times by Northern blot analysis. The figure represents autoradiograms exposed for 24 h at -80 °C. The right part of panel C represents the densitometric scanning of the Northern blots obtained from 7TD1 cells incubated between 6 and 9 h with: IL-6 plus cycloheximide (box) or IL-6 plus cycloheximide in the presence of actinomycin D ().


To investigate a possible combined effect of cycloheximide and actinomycin D on junB mRNA stability, we blocked cellular transcription with actinomycin D, 6 h after the addition of IL-6 and cycloheximide, and measured junB mRNA decay.

As shown in Fig. 7, panel C, we found that the time course of junB mRNA shut-off, determined by Northern blot analysis after the addition of actinomycin D and quantified by densitometric scanning (right part of panel C), was not modified in the presence of cycloheximide, compared to Fig. 5, panel D, confirming that the effect of this drug is consistent with a sole increase of the junB transcription rate and did not lie on increased junB mRNA stability.

In contrast with results obtained on junB mRNA, cycloheximide does not potentiate but rather inhibits c-myc mRNA expression (not shown).

Tyrphostin and Genistein Inhibited, Respectively, the Early- and the Late-induced junB mRNA Wave

As described by others(29, 30) , induction by IL-6 of the early junB mRNA requires activation of tyrosine kinase(s). However, to date, no study has been reported concerning the induction of the late-induced junB mRNA level. In an attempt to clarify this question, we have tested the effect of one of the tyrphostins (25, RG50875) and of genistein, two potent tyrosine kinase inhibitors, on the induction of both early- and late-induced junB mRNA in 7TD1 cells. Thus, 7TD1 cells were preincubated for 30 min with tyrphostin (100 µg/ml) or genistein (50 µg/ml), then IL-6 (100 units/ml) was added and the level of junB mRNA was followed for 1, 3, and 6 h. As expected(29, 30) , tyrphostin 25 inhibited the expression of the early junB mRNA by greater than 90-95%, as shown in Fig. 8, but surprisingly, did not block the stimulation of the late-induced junB expression, suggesting that a tyrphostin-sensitive tyrosine kinase controls the first junB mRNA wave. At variance with results published by Nakajima and Wall (30) , genistein, another tyrosine kinase inhibitor, did not affect the early-induced junB mRNA peak but, interestingly, abolished the expression of the second wave, suggesting that, similarly to the early-induced peak, the activation of the delayed junB mRNA also required activation of a tyrosine kinase.

Figure 8: Effect of tyrosine kinase inhibitors on junB mRNA stimulated by IL-6. Quiescent 7TD1 cells were preincubated for 30 min with either genistein (50 µg/ml) or tyrphostin 25 (100 µg/ml) and stimulated with IL-6 (100 units/ml) for, respectively, 1, 3, and 6 h. Total RNAs were purified, separated on agarose gels, blotted onto nylon membrane, and finally hybridized, as described under ``Materials and Methods,'' with P-labeled cDNA probe encoding for junB (left part). On the right part is shown the level of ribosomal 18 S RNA visualized by ethidium bromide as a control of RNA loading.


Taking together the present results suggested that IL-6 triggered two distinct tyrosine kinase activities that controlled expression of the early- and late-induced junB mRNA, respectively.

DISCUSSION

Interleukin-6 is responsible for various biological effects such as inflammation and growth of myeloma or hybridoma cells. To date, the mechanisms underlying IL-6 effects are a matter of intense research. In an attempt to gain information on molecular events involved in IL-6 mitogenic action, we have used the mouse B hybridoma cell line 7TD1, which is IL-6-dependent for survival and proliferation, and studied the regulation by this cytokine of junB, a cellular oncogene acknowledged to be closely related to the control of cell growth (43, 44, 45) .

junB is a typical early and transiently activated oncogene, inducible by a wide variety of stimuli that elicit diverse biological functions(29, 30, 39, 46, 47, 48) . We report here that, in 7TD1 cells, IL-6 stimulated junB mRNA in a biphasic fashion. The first peak presented a sharply transient aspect while the second rise in junB mRNA was more sustained. The IL-6 effect was specific of junB, since c-myc that was constitutively expressed at high levels in this biological model was not further stimulated in the presence of the cytokine. We demonstrated by the means of run-on experiments that the two peaks in junB mRNA resulted exclusively, in both cases, from an increase in the transcription rate, without any effect on mRNA stability. The induction of the immediate junB mRNA early peak culminated at 30-60 min and rapidly declined to the basal value, by 2 h. It is noteworthy that the declining phase presented the same time course regardless of the presence of IL-6 in the medium. This shut-off mechanism was specific for junB since during the same time span c-myc transcription remained unaffected. Inhibition of protein synthesis by cycloheximide did not modify the kinetic profile of this IL-6-induced early peak of junB mRNA. These data suggest that the start and the arrest of junB transcription during this process were not dependent on the synthesis of new factors, but rather stem from post-translational modifications of factors pre-existing to the addition of the cytokine. The situation observed following the second peak of junB mRNA was different in the sense that, intriguingly, cycloheximide strongly potentiated the IL-6-stimulated junB transcription rate. Thus, our data are in agreement with the occurrence of a constitutive junB transcriptional repressor which specifically regulates the second wave of junB mRNA synthesis. This observation is in accord with a previous report showing a superinduction effect of cycloheximide on junB under fetal calf serum or growth factor, like fibroblast growth factor or platelet-derived growth factor, stimulation on mouse fibroblasts(39, 47) . It is noteworthy that the presence of such a repressor was only detectable during the onset of the second wave of transcription which would mean that its activation requires de novo protein synthesis occurring after the first peak of junB mRNA. As demonstrated by the experiments using actinomycin D, the prolonged expression of junB mRNA during the second phase was due to a sustained transcription activity that was itself strictly dependent on the permanent presence of IL-6. In contrast with the first peak's features, no deactivation in transcriptional activity was observed during the second wave of transcripts, provided that IL-6 was present in the medium. Furthermore, the declining phase that occurred after 8-9 h did not result from an IL-6-driven deactivation mechanism, but rather reflected the fact that cells entered into S phase during which transcriptional activity is largely decreased. In fact, addition of aphidicoline that impedes cells to reinitiate their DNA replication program maintained the maximal level of junB transcripts during the second peak, all along the time this drug was present in the incubation medium (not shown).

Recent advances have demonstrated that the early induction of junB correlates with phosphorylation of a p160 protein by a still unidentified tyrosine kinase(29, 30) . In this report we found that two kinds of protein tyrosine kinases were involved in the complex control of junB under IL-6 stimulation. A tyrphostin-sensitive tyrosine kinase controlled the onset of the early transient expression of junB, whereas a genistein-sensitive tyrosine kinase was involved in the control of the more sustained second peak of junB mRNA. Owing to the discriminative action of the tyrosine kinase inhibitors, we may postulate that two distinct pathways regulate the expression of the two waves of junB, that are each under the control of a defined tyrosine kinase. The discriminatory effect we observed on 7TD1 cells between these two compounds is not an original feature. For example, on NIH3T3 cells, tyrphostin (RG50864) and genistein exert discriminative inhibitory effects on the epidermal growth factor-stimulated pathways leading to the activation of either microtubule associated protein kinase (49) or p70S6 kinase(50) .

Likewise, discriminative inhibitory effects among tyrphostin congeners have been reported for epidermal growth factor receptor (HER1) and neu/ErB2 (HER2) which share 80% sequence homology in the kinase domain (51) . Tyrphostins can also discriminate between p140, p185, and p210in vitro although these receptor species only differ in their NH(2)-terminal sequence (52) . Since such a discrepancy exists among tyrphostins, it is likely to expect that differences might exist between the action of genistein and tyrphostin. In this respect, combination of different tyrosine kinase inhibitors might be useful to dissect different aspects of the IL-6 response.

Our data give clues to investigate whether the repressor factor that negatively regulated the increase in transcription rate of the second peak was dependent of the occurrence of the first peak of junB mRNA. Indeed, the pattern of expression of the second wave of transcripts was not significantly modified in the presence of tyrphostin that abolished the first junB mRNA peak. One can thus conclude that the synthesis of a repressor factor, acting during the second peak, was not dependent on JunB protein molecules that would have been encoded by early junB transcripts. Taken collectively, these data highly suggest that the regulation of the two waves of junB are two separate events controlled by two distinct pathways.

Recently, the involvement of Jak congeners in the signal transduction mediated by IL-6 effects has been clearly established on different cell types(16, 17, 53) . We are currently investigating a possible involvement of this family of tyrosine kinases in our model, especially in the mediation of junB expression.

Owing to the complex control that IL-6 exerts on junB mRNA induction, we wished to know what was the fate of the JunB protein in the presence of the cytokine. Interestingly, JunB protein was undetectable in nucleic fractions purified from nonstimulated 7TD1 cells but was strongly enhanced in presence of IL-6. As assessed by Western blotting experiments, the protein increased in a biphasic fashion that matched the biphasic increase of mRNA and the transcription rate. The JunB protein encoded by the two waves of mRNA migrated as a monomer of 46 kDa which might correspond to the molecular mass of a phosphorylated form of the molecule, as compared with the migration pattern of the 39-kDa unphosphorylated recombinant protein. This result then supposes that IL-6 not only increased the nuclear level of JunB but could also stimulate the phosphorylation of this protein. On a mechanistic point of view, this observation is interesting. In fact, it is now well accepted that the phosphorylation status of oncogene-encoded proteins like c-Fos, c-Jun, or JunB might control their binding activity(48, 54, 55, 56) . Experiments using recombinant JunB protein are currently in progress, aimed at clarifying the physiological role of IL-6-dependent phosphorylation on JunB binding and transactivating activities in 7TD1 cells.

To date, the main interest in the field of IL-6 action has focused on the characterization of intermediates involved in the generation and the propagation of early signals triggered by this cytokine. Using junB oncogene as a molecular reporter, we could demonstrate that, besides its early action, IL-6 also stimulates mid/long term events. By the use of cycloheximide and tyrosine kinase inhibitors we were able to reveal two distinct signaling routes that end up with the expression of a same molecular event, namely JunB expression. Indeed, the control of the two peaks of junB mRNA differs on three major points: (i) activation of a repressor protein that intervened only in the control of the transcription rate occurring during the second peak, (ii) the presence of IL-6 impeded the decrease in the second burst of transcription rate whereas the presence of the cytokine had no effect on the onset of the declining phase of the first peak, (iii) tyrosine kinase inhibitors allowed us to hypothesize that two distinct protein tyrosine kinases control each of the two peaks of IL-6-induced junB transcripts.

In conclusion, our data provide interesting tools to approach the mechanisms by which junB could participate in the mediation of IL-6 biological effects.

FOOTNOTES

*
This work was supported by the Institut National de la Recherche Médicale (INSERM) and Grant 6717 from the Association pour la Recherche contre le Cancer (ARC). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 33-93-37-77-02; Fax: 33-93-81-94-56.

Present address: Oncogenic Viruses Unit, Dept. of Biotechnology, Pasteur Institut, 25, Rue du Dr. Roux, 75724 Paris Cedex 15, France.

**
Present address: Glaxo Institute for Molecular Biology S.A., 14, Chemin des Aulx, 1228 Plan-les-Ouates/Geneva, Switzerland.

(^1)
The abbreviations used are: IL-6, interleukin-6; genistein, 4`,5,7-trihydroxyisoflavone; tyrphostin 25 (RG50875), (3,4,5-trihydroxybenzilidene)-malononitrile; NF-IL-6, nuclear factor IL-6; MOPS, 4-morpholinepropanesulfonic acid.

ACKNOWLEDGEMENTS

We want to express our sincere thanks to Dr. J. Van Snick (Brussel) for kindly providing the 7TD1 cell line. We also thank Dr. M. Yaniv (Institut Pasteur, Paris) for generously giving the specific anti-junB antibody (JunB 2.2). We are indebted to R. Mescatullo for illustration work.

REFERENCES

  1. Andus, T., Geiger, T., Hirano, T., Northoff, H., Ganter, U., Bauer, J., Kishimoto, T., and Heinrich, P. C. (1987) FEBS Lett. 221, 18-22 [CrossRef][Medline] [Order article via Infotrieve]
  2. Gauldie, J., Richards, C., Harnish, D., Landsdorp, P., and Baumann, H. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 7251-7255 [Abstract/Free Full Text]
  3. Hirano, T., Taga, T., Nakano, N., Yasukawa, K., Kashiwamura, S., Shimizu, S., Nakajima, K., Pyun, K. H., and Kishimoto, T. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 5490-5494 [Abstract/Free Full Text]
  4. Muragushi, A., Hirano, T., Tang, B., Matsuda, T., Horii, Y., Nakajima, K., and Kishimoto, T. (1988) J. Exp. Med. 167, 332-344 [Abstract/Free Full Text]
  5. Hama, T., Miyamoto, M., Tsukui, H., Nishio, C., and Hatanaka, H. (1989) Neurosci. Lett. 104, 340-344 [CrossRef][Medline] [Order article via Infotrieve]
  6. Miyaura, C., Onozaki, K., Akiyama, Y., Taniyama, T., Hirano, T., Kishimoto, T., and Suda, T. (1988) FEBS Lett. 234, 17-21 [CrossRef][Medline] [Order article via Infotrieve]
  7. Satoh, T., Nakamura, S., Taga, T., Matsuda, T., Hirano, T., Kishimoto, T., and Kaziro, T. (1988) Mol. Cell. Biol. 8, 3546-3549 [Abstract/Free Full Text]
  8. Takai, Y., Wong, G. G., Clark, S., Burakoff, S., and Herrmann, S. (1988) J. Immunol. 140, 508-512 [Abstract]
  9. Nordan, R. P., and Potter, M. (1986) Science 233, 566-569 [Abstract/Free Full Text]
  10. Uyttenhove, C., Coulie, P. G., and Van Snick, J. (1988) J. Exp. Med. 167, 1417-1427 [Abstract/Free Full Text]
  11. Van Damme, J., Opdenakker, G., Simpson, R. J., Rubira, M. R., Cayphas, S., Vink, A., Billiau, A., and Van Snick, J. (1987) J. Exp. Med. 165, 914-919 [Abstract/Free Full Text]
  12. Kawano, M., Hirano, T., Matsuda, T., Horii, Y., Iwato, H., Asaoku, H., Tang, B., Tanaka, H., Kuramoto, A., and Kishimoto, T. (1988) Nature 332, 83-85 [CrossRef][Medline] [Order article via Infotrieve]
  13. Murakami, M., Narazaki, M., Hibi, M., Yawata, H., Yasukawa, K., Hamaguchi, M., Taga, T., and Kishimoto, T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11349-11353 [Abstract/Free Full Text]
  14. Taga, T., Hibi, M., Hirata, Y., Yamasaki, K., Yasukawa, K., Matsuda, T., Hirano, T., and Kishimoto, T. (1989) Cell 58, 573-581 [CrossRef][Medline] [Order article via Infotrieve]
  15. Murakami, M., Hibi, M., Nakagawa, N., Nakagawa, T., Yasukawa, K., Yamanishi, K., Taga, T., and Kishimoto, T. (1993) Science 260, 1808-1810 [Abstract/Free Full Text]
  16. Lütticken, C., Wegenka, U. M., Yuan, J., Buschmann, J., Schindler, C., Ziemiecki, A., Harpur, A. G., Wilks, A. F., Yasukawa, K., Taga, T., Kishimoto, T., Barbieri, G., Pellegrini, S., Sendtner, M., Heinrich, P. C., and Horn, F. (1994) Science 263, 89-92 [Abstract/Free Full Text]
  17. Stahl, N., Boulton, T. G., Farruggella, T., Ip, N. Y., Davis, S., Witthuhn, B. A., Quelle, F. W., Silvennoinen, O., Barbieri, G., Pellegrini, S., Ihle, J. N., and Yancopoulos, G. D. (1994) Science 263, 92-95 [Abstract/Free Full Text]
  18. Hattori, M., Abraham, L. J., Northemann, W., and Fey, G. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2364-2368 [Abstract/Free Full Text]
  19. Hocke, G. M., Barry, D., and Fey, G. H. (1992) Mol. Cell. Biol. 12, 2282-2294 [Abstract/Free Full Text]
  20. Wegenka, U. M., Buschmann, J., Lütticken, C., Heinrich, P. C., and Horn, F. (1993) Mol. Cell. Biol. 13, 276-288 [Abstract/Free Full Text]
  21. Melamed, D., Resnitzky, D., Haimov, I., Levy, N., Pfarr, C. M., Yaniv, M., and Kimchi, A. (1993) Cell Growth Differ. 4, 689-698 [Abstract]
  22. Melamed, D., Tiefenbrun, N., Yarden, A., and Kimchi, A. (1993) Mol. Cell. Biol. 13, 5255-5265 [Abstract/Free Full Text]
  23. Poli, V., Mancini, F. P., and Cortese, R. (1990) Cell 63, 643-653 [CrossRef][Medline] [Order article via Infotrieve]
  24. Harroch, S., Revel, M., and Chebath, J. (1994) EMBO J. 13, 1942-1949 [Medline] [Order article via Infotrieve]
  25. Zhong, Z., Wen, Z., and Darnell, J. E., Jr. (1994) Science 264, 95-98 [Abstract/Free Full Text]
  26. Akira, S., Isshiki, H., Sugita, T., Tanabe, O., Kinoshita, S., Nishio, Y., Nakajima, T., Hirano, T., and Kishimoto, T. (1990) EMBO J. 9, 1897-1906 [Medline] [Order article via Infotrieve]
  27. Nakajima, T., Kinoshita, S., Sasagawa, T., Sasaki, K., Naruto, M., Kishimoto, T., and Akira, S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2207-2211 [Abstract/Free Full Text]
  28. Wegenka, U. M., Lütticken, C., Buschmann, J., Yuan, J., Lottspeich, F., Müller-Esterl, W., Schindler, C., Roeb, E., Heinrich, P. C., and Horn, F. (1994) Mol. Cell. Biol. 14, 3186-3196 [Abstract/Free Full Text]
  29. Lord, K. A., Abdollahi, A., Thomas, S. M., De Marco, M., Brugge, J. S., Hoffman-Liebermann, B., and Liebermann, D. A. (1991) Mol. Cell. Biol. 11, 4371-4379 [Abstract/Free Full Text]
  30. Nakajima, K., and Wall, R. (1991) Mol. Cell. Biol. 11, 1409-1418 [Abstract/Free Full Text]
  31. Vogt, P. K., and Bos, T. J. (1990) Adv. Cancer Res. 55, 1-35 [Medline] [Order article via Infotrieve]
  32. Boise, L. H., Petryniak, B., Mao, X., June, C. H., Wang, C. Y., Lindsten, T., Bravo, R., Kovary, K., Leiden, J. M., and Thompson, C. B. (1993) Mol. Cell. Biol. 13, 1911-1919 [Abstract/Free Full Text]
  33. Nakajima, K., Kusafuka, T., Takeda, T., Fujitani, Y., Nakae, K., and Hirano, T. (1993) Mol. Cell. Biol. 13, 3027-3041 [Abstract/Free Full Text]
  34. Proudfoot, A. E. I., Brown, S. C., Bernard, A. R., Bonnefoy, J. Y., and Kawashima, E. H. (1993) J. Protein Chem. 12, 489-497 [CrossRef][Medline] [Order article via Infotrieve]
  35. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18, 5294-5299 [CrossRef][Medline] [Order article via Infotrieve]
  36. Ryder, K., Lau, L. F., and Nathan, D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1487-1491 [Abstract/Free Full Text]
  37. Doglio, A., Dani, C., Grimaldi, P., and Ailhaud, G. (1986) Biochem. J. 238, 123-129 [Medline] [Order article via Infotrieve]
  38. Dignam, J. D., Lebovits, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475-1479 [Abstract/Free Full Text]
  39. Ryder, K., and Nathans, D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8464-8467 [Abstract/Free Full Text]
  40. Trejo, J., Chambard, J. C., Karin, M., and Brown, J. H. (1992) Mol. Cell. Biol. 12, 4742-4750 [Abstract/Free Full Text]
  41. Carter, R., Cosenza, S. C., Pena, A., Lipson, K., Soprano, D. R., and Soprano, K. J. (1991) Oncogene 6, 229-235 [Medline] [Order article via Infotrieve]
  42. Pulverer, B. J., Hugues, K., Franklin, C. C., Kraft, A. S., Leevers, S. J., and Woodgett, J. R. (1993) Oncogene 8, 407-415 [Medline] [Order article via Infotrieve]
  43. Kovary, K., and Bravo, R. (1991) Mol. Cell. Biol. 11, 4466-4472 [Abstract/Free Full Text]
  44. Schütte, J., Viallet, J., Nau, M., Segal, S., Fedorko, J., and Minna, J. (1989) Cell 59, 987-997 [CrossRef][Medline] [Order article via Infotrieve]
  45. Castellazzi, M., Spyrou, G., La Vista, N., Dangy, J. P., Piu, F., Yaniv, M., and Brun, G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8890-8894 [Abstract/Free Full Text]
  46. De Groot, R. P., Auwerx, J., Karperien, M., Staels, B., and Kruijer, W. (1991) Nucleic Acids Res. 19, 775-780 [Abstract/Free Full Text]
  47. Kitabayashi, I., Kawakami, Z., Matsuoka, T., Chiu, R., Gachelin, G., and Yokoyama, K. (1993) J. Biol. Chem. 268, 14482-14489 [Abstract/Free Full Text]
  48. Kovary, K., and Bravo, R. (1991) Mol. Cell. Biol. 11, 2451-2459 [Abstract/Free Full Text]
  49. Agbotounou, W. K., Mousset, S., Piperno, S., Pierre, M., Jacquemin-Sablon, A., and Pierre, J. (1994) Biochem. Pharmacol. 48, 505-515 [CrossRef][Medline] [Order article via Infotrieve]
  50. Linassier, C., Pierre, M., Le Pecq, J. B., and Pierre, J. (1990) Biochem. Pharmacol. 39, 187-193 [CrossRef][Medline] [Order article via Infotrieve]
  51. Levitzky, A. (1992) FASEB J. 6, 3275-3282 [Abstract]
  52. Anafi, M., Gazit, A., Gilon, C., Ben-Neriah, Y., and Levitzki, A. (1992) J. Biol. Chem. 267, 4518-4523 [Abstract/Free Full Text]
  53. Narazaki, M., Witthuhn, B. A., Yoshida, K., Silvennoinen, O., Yasukawa, K., Ihle, J. N., Kishimoto, T., and Taga, T. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2285-2289 [Abstract/Free Full Text]
  54. Binetruy, B., Smeal, T., and Karin, M. (1991) Nature 351, 122-127 [CrossRef][Medline] [Order article via Infotrieve]
  55. Boyle, W. J., Smeal, T., Defize, L. H. K., Angel, P., Woodgett, J. R., Karin, M., and Hunter, T. (1991) Cell 64, 573-584 [CrossRef][Medline] [Order article via Infotrieve]
  56. Offir, R., Dwarki, V. J., Rashid, D., and Verma, I. M. (1990) Nature 348, 78-80
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

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