FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2070333003> ?p ?o ?g. }

• W2070333003 endingPage "1268" @default.
• W2070333003 startingPage "1261" @default.
• W2070333003 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. 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. INTRODUCTIONInterleukin-6 (IL-6) 1The abbreviations used are: IL-6interleukin-6genistein4′,5,7-trihydroxyisoflavonetyrphostin 25 (RG50875)(3,4,5-trihydroxybenzilidene)-malononitrileNF-IL-6nuclear factor IL-6MOPS4-morpholinepropanesulfonic acid. 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(1Andus T. Geiger T. Hirano T. Northoff H. Ganter U. Bauer J. Kishimoto T. Heinrich P.C. FEBS Lett. 1987; 221: 18-22Crossref PubMed Scopus (244) Google Scholar, 2Gauldie J. Richards C. Harnish D. Landsdorp P. Baumann H. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7251-7255Crossref PubMed Scopus (1358) Google Scholar, 3Hirano T. Taga T. Nakano N. Yasukawa K. Kashiwamura S. Shimizu S. Nakajima K. Pyun K.H. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5490-5494Crossref PubMed Scopus (563) Google Scholar, 4Muragushi A. Hirano T. Tang B. Matsuda T. Horii Y. Nakajima K. Kishimoto T. J. Exp. Med. 1988; 167: 332-344Crossref PubMed Scopus (630) Google Scholar), T cell activation, and neuronal cell and macrophage differentiation(5Hama T. Miyamoto M. Tsukui H. Nishio C. Hatanaka H. Neurosci. Lett. 1989; 104: 340-344Crossref PubMed Scopus (244) Google Scholar, 6Miyaura C. Onozaki K. Akiyama Y. Taniyama T. Hirano T. Kishimoto T. Suda T. FEBS Lett. 1988; 234: 17-21Crossref PubMed Scopus (127) Google Scholar, 7Satoh T. Nakamura S. Taga T. Matsuda T. Hirano T. Kishimoto T. Kaziro T. Mol. Cell. Biol. 1988; 8: 3546-3549Crossref PubMed Scopus (373) Google Scholar, 8Takai Y. Wong G.G. Clark S. Burakoff S. Herrmann S. J. Immunol. 1988; 140: 508-512PubMed Google Scholar). It has also been described to promote the growth of some murine hybridoma and plasmocytoma(9Nordan R.P. Potter M. Science. 1986; 233: 566-569Crossref PubMed Scopus (281) Google Scholar, 10Uyttenhove C. Coulie P.G. Van Snick J. J. Exp. Med. 1988; 167: 1417-1427Crossref PubMed Scopus (205) Google Scholar, 11Van Damme J. Opdenakker G. Simpson R.J. Rubira M.R. Cayphas S. Vink A. Billiau A. Van Snick J. J. Exp. Med. 1987; 165: 914-919Crossref PubMed Scopus (537) Google Scholar), as well as human myeloma(12Kawano M. Hirano T. Matsuda T. Horii Y. Iwato H. Asaoku H. Tang B. Tanaka H. Kuramoto A. Kishimoto T. Nature. 1988; 332: 83-85Crossref PubMed Scopus (1444) Google Scholar). 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 (13Murakami M. Narazaki M. Hibi M. Yawata H. Yasukawa K. Hamaguchi M. Taga T. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11349-11353Crossref PubMed Scopus (486) Google Scholar, 14Taga T. Hibi M. Hirata Y. Yamasaki K. Yasukawa K. Matsuda T. Hirano T. Kishimoto T. Cell. 1989; 58: 573-581Abstract Full Text PDF PubMed Scopus (1186) Google Scholar). 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)(15Murakami M. Hibi M. Nakagawa N. Nakagawa T. Yasukawa K. Yamanishi K. Taga T. Kishimoto T. Science. 1993; 260: 1808-1810Crossref PubMed Scopus (638) Google Scholar, 16Lütticken 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. Horn F. Science. 1994; 263: 89-92Crossref PubMed Scopus (704) Google Scholar, 17Stahl 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. Yancopoulos G.D. Science. 1994; 263: 92-95Crossref PubMed Scopus (840) Google Scholar).In response to IL-6, several transcription factors are activated (18Hattori M. Abraham L.J. Northemann W. Fey G.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2364-2368Crossref PubMed Scopus (143) Google Scholar, 19Hocke G.M. Barry D. Fey G.H. Mol. Cell. Biol. 1992; 12: 2282-2294Crossref PubMed Google Scholar, 20Wegenka U.M. Buschmann J. Lütticken C. Heinrich P.C. Horn F. Mol. Cell. Biol. 1993; 13: 276-288Crossref PubMed Scopus (485) Google Scholar, 21Melamed D. Resnitzky D. Haimov I. Levy N. Pfarr C.M. Yaniv M. Kimchi A. Cell Growth Differ. 1993; 4: 689-698PubMed Google Scholar, 22Melamed D. Tiefenbrun N. Yarden A. Kimchi A. Mol. Cell. Biol. 1993; 13: 5255-5265Crossref PubMed Scopus (67) Google Scholar, 23Poli V. Mancini F.P. Cortese R. Cell. 1990; 63: 643-653Abstract Full Text PDF PubMed Scopus (455) Google Scholar, 24Harroch S. Revel M. Chebath J. EMBO J. 1994; 13: 1942-1949Crossref PubMed Scopus (81) Google Scholar, 25Zhong Z. Wen Z. Darnell Jr., J.E. Science. 1994; 264: 95-98Crossref PubMed Scopus (1691) Google Scholar). 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/EBPβ) was initially identified as a factor that binds to the AGATTGCACAATCT consensus sequence encompassed within the IL-6 promoter(26Akira S. Isshiki H. Sugita T. Tanabe O. Kinoshita S. Nishio Y. Nakajima T. Hirano T. Kishimoto T. EMBO J. 1990; 9: 1897-1906Crossref PubMed Scopus (1203) Google Scholar). This factor was involved in the induction of several class 1 acute phase protein genes (α1-acid glycoprotein, angiotensinogen …) by IL-6(23Poli V. Mancini F.P. Cortese R. Cell. 1990; 63: 643-653Abstract Full Text PDF PubMed Scopus (455) Google Scholar). 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)(27Nakajima T. Kinoshita S. Sasagawa T. Sasaki K. Naruto M. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2207-2211Crossref PubMed Scopus (513) Google Scholar). 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 (α2-macroglobulin, α1-antichymotrypsin …). They have been identified as IL-6 response element binding protein (18Hattori M. Abraham L.J. Northemann W. Fey G.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2364-2368Crossref PubMed Scopus (143) Google Scholar, 19Hocke G.M. Barry D. Fey G.H. Mol. Cell. Biol. 1992; 12: 2282-2294Crossref PubMed Google Scholar) and acute phase response factor/stat-3(20Wegenka U.M. Buschmann J. Lütticken C. Heinrich P.C. Horn F. Mol. Cell. Biol. 1993; 13: 276-288Crossref PubMed Scopus (485) Google Scholar, 25Zhong Z. Wen Z. Darnell Jr., J.E. Science. 1994; 264: 95-98Crossref PubMed Scopus (1691) Google Scholar, 28Wegenka U.M. Lütticken C. Buschmann J. Yuan J. Lottspeich F. Müller-Esterl W. Schindler C. Roeb E. Heinrich P.C. Horn F. Mol. Cell. Biol. 1994; 14: 3186-3196Crossref PubMed Scopus (232) Google Scholar). 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(19Hocke G.M. Barry D. Fey G.H. Mol. Cell. Biol. 1992; 12: 2282-2294Crossref PubMed Google Scholar). 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 (16Lütticken 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. Horn F. Science. 1994; 263: 89-92Crossref PubMed Scopus (704) Google Scholar).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(29Lord K.A. Abdollahi A. Thomas S.M. De Marco M. Brugge J.S. Hoffman-Liebermann B. Liebermann D.A. Mol. Cell. Biol. 1991; 11: 4371-4379Crossref PubMed Scopus (146) Google Scholar, 30Nakajima K. Wall R. Mol. Cell. Biol. 1991; 11: 1409-1418Crossref PubMed Scopus (115) Google Scholar), one possible component of the transcription factors AP-1 (31Vogt P.K. Bos T.J. Adv. Cancer Res. 1990; 55: 1-35Crossref PubMed Scopus (283) Google Scholar) or NFAT(32Boise L.H. Petryniak B. Mao X. June C.H. Wang C.Y. Lindsten T. Bravo R. Kovary K. Leiden J.M. Thompson C.B. Mol. Cell. Biol. 1993; 13: 1911-1919Crossref PubMed Scopus (209) Google Scholar). 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(33Nakajima K. Kusafuka T. Takeda T. Fujitani Y. Nakae K. Hirano T. Mol. Cell. Biol. 1993; 13: 3027-3041Crossref PubMed Scopus (114) Google Scholar). This factor is activated by a H7-sensitive pathway that does not involved protein kinase C, protein kinase A, or microtubule associated protein kinases(33Nakajima K. Kusafuka T. Takeda T. Fujitani Y. Nakae K. Hirano T. Mol. Cell. Biol. 1993; 13: 3027-3041Crossref PubMed Scopus (114) Google Scholar), 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 METHODSReagentsRecombinant human interleukin-6 (IL-6), purified as described elsewhere(34Proudfoot A.E.I. Brown S.C. Bernard A.R. Bonnefoy J.Y. Kawashima E.H. J. Protein Chem. 1993; 12: 489-497Crossref PubMed Scopus (19) Google Scholar), 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 [α-32P]dCTP (3000 Ci/mmol) and [α-32P]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-β-D-galactopyranoside, and glutathione-Sepharose 4B were from Pharmacia/LKB (St-Quentin Yvelines, France).Cell Culture7TD1 cells (2.5 to 3.5 × 105/ml) were maintained at 37°C, in 8% CO2 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 AnalysisTotal cellular RNA was prepared by denaturation in guanidinium thiocyanate followed by pelleting through a cesium chloride cushion(35Chirgwin J.M. Przybyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16619) Google Scholar). 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 (106 cpm/ml) were carried out overnight at 65°C in a QuickHyb solution (Stratagene), filters were washed in 0.1 × 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 ProbesThe 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(36Ryder K. Lau L.F. Nathan D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1487-1491Crossref PubMed Scopus (513) Google Scholar). The murine c-myc cDNA was a generous gift from Dr. Dani (Nice, France).Nuclei Isolation and Run-on Transcription AssayNuclei were isolated from 7TD1 cells (2-4 × 107) and run-on transcription assay performed according to the method described by Doglio et al.(37Doglio A. Dani C. Grimaldi P. Ailhaud G. Biochem. J. 1986; 238: 123-129Crossref PubMed Scopus (92) Google Scholar). 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 107 cpm of biosynthetically 32P-labeled nuclear RNAs, then washed four times at 65°C for 2 h with 2 × SSC, 0.1% SDS. Filters were dried and exposed to Amersham Hyperfilms MP at −80°C.Preparation of Nuclear Extracts and Immunoblot AnalysisNuclear extracts were prepared using a method derived from Dignam et al.(38Dignam J.D. Lebovits R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1479Crossref PubMed Scopus (9142) Google Scholar). Briefly, nuclei (4-5 107) 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 MgCl2, 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 ImmunoblottingThis 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-β-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.RESULTSJunB mRNA Is Stimulated in a Biphasic Fashion in Response to IL-6 on 7TD1 CellsThe jun family members junB and c-jun are usually transiently stimulated in the early response to numerous stimuli including IL-6(29Lord K.A. Abdollahi A. Thomas S.M. De Marco M. Brugge J.S. Hoffman-Liebermann B. Liebermann D.A. Mol. Cell. Biol. 1991; 11: 4371-4379Crossref PubMed Scopus (146) Google Scholar, 30Nakajima K. Wall R. Mol. Cell. Biol. 1991; 11: 1409-1418Crossref PubMed Scopus (115) Google Scholar, 39Ryder K. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8464-8467Crossref PubMed Scopus (340) Google Scholar). 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(40Trejo J. Chambard J.C. Karin M. Brown J.H. Mol. Cell. Biol. 1992; 12: 4742-4750Crossref PubMed Scopus (60) Google Scholar, 41Carter R. Cosenza S.C. Pena A. Lipson K. Soprano D.R. Soprano K.J. Oncogene. 1991; 6: 229-235PubMed Google Scholar).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).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 ProteinTo 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)It is currently accepted that the phosphorylation of c-Jun is responsible for its electrophoretic retardation from 39 kDa to 46 kDa (42Pulverer B.J. Hugues K. Franklin C.C. Kraft A.S. Leevers S.J. Woodgett J.R. Oncogene. 1993; 8: 407-415PubMed Google Scholar). 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 MannerTo 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 [α-32P]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 (30Nakajima K. Wall R. Mol. Cell. Biol. 1991; 11: 1409-1418Crossref PubMed Scopus (115) Google Scholar), 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 [α-32P]UTP was incorporated into nascent RNA chains as described under “Materials and Methods.” Labeled RNA (107 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 (□) and c-myc (■) cDNAs, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)IL-6 Does Not Modify the Stability of junB mRNAIn 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 Figure 4:, Figure 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 □, ■, ○, and •.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 □, ■, ○, and • correspond, respectively, to panels B, C, D, and E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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-lif" @default.
• W2070333003 created "2016-06-24" @default.
• W2070333003 creator A5005836091 @default.
• W2070333003 creator A5007874461 @default.
• W2070333003 creator A5019109478 @default.
• W2070333003 creator A5027509313 @default.
• W2070333003 creator A5044014386 @default.
• W2070333003 creator A5059732459 @default.
• W2070333003 date "1995-01-01" @default.
• W2070333003 modified "2023-10-15" @default.
• W2070333003 title "Two Distinct Signalling Pathways Are Involved in the Control of the Biphasic junB Transcription Induced by Interleukin-6 in the B Cell Hybridoma 7TD1" @default.
• W2070333003 cites W11800597 @default.
• W2070333003 cites W126627744 @default.
• W2070333003 cites W1511577045 @default.
• W2070333003 cites W1558484175 @default.
• W2070333003 cites W179818011 @default.
• W2070333003 cites W1811606715 @default.
• W2070333003 cites W1822742523 @default.
• W2070333003 cites W1884284628 @default.
• W2070333003 cites W1904166955 @default.
• W2070333003 cites W1954706670 @default.
• W2070333003 cites W1964842789 @default.
• W2070333003 cites W1967653587 @default.
• W2070333003 cites W1967761338 @default.
• W2070333003 cites W1971615288 @default.
• W2070333003 cites W1971908211 @default.
• W2070333003 cites W1975773833 @default.
• W2070333003 cites W1979655386 @default.
• W2070333003 cites W1981799488 @default.
• W2070333003 cites W1984361597 @default.
• W2070333003 cites W1993243592 @default.
• W2070333003 cites W1994975139 @default.
• W2070333003 cites W1996548067 @default.
• W2070333003 cites W1999028040 @default.
• W2070333003 cites W2007000374 @default.
• W2070333003 cites W2020211894 @default.
• W2070333003 cites W2026066120 @default.
• W2070333003 cites W2029526969 @default.
• W2070333003 cites W2031451107 @default.
• W2070333003 cites W2036452124 @default.
• W2070333003 cites W2037540469 @default.
• W2070333003 cites W2040020188 @default.
• W2070333003 cites W2042917826 @default.
• W2070333003 cites W2043590802 @default.
• W2070333003 cites W2060333964 @default.
• W2070333003 cites W2063281625 @default.
• W2070333003 cites W2081841050 @default.
• W2070333003 cites W2094111001 @default.
• W2070333003 cites W2096746473 @default.
• W2070333003 cites W2112916314 @default.
• W2070333003 cites W2115798976 @default.
• W2070333003 cites W2122048408 @default.
• W2070333003 cites W2126440278 @default.
• W2070333003 cites W2166788322 @default.
• W2070333003 cites W2168822788 @default.
• W2070333003 cites W2170261399 @default.
• W2070333003 cites W2230705365 @default.
• W2070333003 cites W30218241 @default.
• W2070333003 cites W4232636542 @default.
• W2070333003 cites W4235134184 @default.
• W2070333003 cites W4240609540 @default.
• W2070333003 cites W4246285205 @default.
• W2070333003 doi "https://doi.org/10.1074/jbc.270.3.1261" @default.
• W2070333003 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7836389" @default.
• W2070333003 hasPublicationYear "1995" @default.
• W2070333003 type Work @default.
• W2070333003 sameAs 2070333003 @default.
• W2070333003 citedByCount "19" @default.
• W2070333003 countsByYear W20703330032014 @default.
• W2070333003 countsByYear W20703330032015 @default.
• W2070333003 crossrefType "journal-article" @default.
• W2070333003 hasAuthorship W2070333003A5005836091 @default.
• W2070333003 hasAuthorship W2070333003A5007874461 @default.
• W2070333003 hasAuthorship W2070333003A5019109478 @default.
• W2070333003 hasAuthorship W2070333003A5027509313 @default.
• W2070333003 hasAuthorship W2070333003A5044014386 @default.
• W2070333003 hasAuthorship W2070333003A5059732459 @default.
• W2070333003 hasBestOaLocation W20703330031 @default.
• W2070333003 hasConcept C104317684 @default.
• W2070333003 hasConcept C138885662 @default.
• W2070333003 hasConcept C179926584 @default.
• W2070333003 hasConcept C185592680 @default.
• W2070333003 hasConcept C2781085546 @default.
• W2070333003 hasConcept C3018449070 @default.
• W2070333003 hasConcept C39743133 @default.
• W2070333003 hasConcept C41895202 @default.
• W2070333003 hasConcept C55493867 @default.
• W2070333003 hasConcept C62478195 @default.
• W2070333003 hasConcept C86339819 @default.
• W2070333003 hasConcept C86803240 @default.
• W2070333003 hasConcept C88498014 @default.
• W2070333003 hasConcept C95444343 @default.
• W2070333003 hasConceptScore W2070333003C104317684 @default.
• W2070333003 hasConceptScore W2070333003C138885662 @default.
• W2070333003 hasConceptScore W2070333003C179926584 @default.
• W2070333003 hasConceptScore W2070333003C185592680 @default.
• W2070333003 hasConceptScore W2070333003C2781085546 @default.
• W2070333003 hasConceptScore W2070333003C3018449070 @default.

• next