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STAT3 is required for the gp130-mediated full activation of the c-myc gene.

Kiuchi N, Nakajima K, Ichiba M, Fukada T, Narimatsu M, Mizuno K, Hibi M, Hirano T - J. Exp. Med. (1999)

Bottom Line: STAT3 does so most likely by binding to cis-regulatory region(s) of the c-myc gene.We show that STAT3 binds to a region overlapping with the E2F site in the c-myc promoter and this site is critical for the c-myc gene promoter- driven transcriptional activation by IL-6 or gp130 signals.This is the first identification of the linkage between a member of the STAT family and the c-myc gene activation, and also explains how the IL-6 family of cytokines is capable of inducing the expression of the c-myc gene.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Oncology, Biomedical Research Center, Osaka University Medical School, Suita, Osaka 565-0871, Japan.

ABSTRACT
The signal transducers and activators of transcription (STAT) family members have been implicated in regulating the growth, differentiation, and death of normal and transformed cells in response to either extracellular stimuli, including cytokines and growth factors, or intracellular tyrosine kinases. c-myc expression is coordinately regulated by multiple signals in these diverse cellular responses. We show that STAT3 mostly mediates the rapid activation of the c-myc gene upon stimulation of the interleukin (IL)-6 receptor or gp130, a signal transducing subunit of the receptor complexes for the IL-6 cytokine family. STAT3 does so most likely by binding to cis-regulatory region(s) of the c-myc gene. We show that STAT3 binds to a region overlapping with the E2F site in the c-myc promoter and this site is critical for the c-myc gene promoter- driven transcriptional activation by IL-6 or gp130 signals. This is the first identification of the linkage between a member of the STAT family and the c-myc gene activation, and also explains how the IL-6 family of cytokines is capable of inducing the expression of the c-myc gene.

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Related in: MedlinePlus

Induction of c-myc  mRNA expression in BAF transformants expressing various  G-CSFR–gp130 chimeric receptors. (A) FACS® analysis for the  expression levels of the chimeric  receptors on the BAF transformants. Cell lines tested were as follows: BAF-G277, BAF/B03 transformants  bearing a chimeric receptor, the extracellular domain of the G-CSFR and  the truncated gp130 including the transmembrane and cytoplasmic 133– amino acid residues, referred to as G133 (shown as BAF-G133), G133F2  with a tyrosine (Y) to phenylalanine (F) mutation at the second tyrosine,  Y759 (BAF-G133F2), G-133F3, with a Y to F mutation at the third  tyrosine Y767 (BAF-G133F3), and G-68, bearing the 68 membrane-proximal amino acid residues of the cytoplasmic region of gp130 (BAF-G68). (B) Northern blot analysis for c-myc mRNA expression in BAF  transformants expressing various G-CSFR–gp130 chimeric receptors.  Total RNAs extracted from the various BAF transformants which had  been deprived of IL-3 for 12 h and stimulated with nothing or G-CSF at  100 ng/ml for the indicated periods of time were subjected to Northern  blot analysis for c-myc mRNA expression. Cell lines tested were as follows: BAF-G277 (lanes 1–3), BAF-G133 (lanes 4–6), BAF-G133F2 (lanes  7–9), G-133F3 (lanes 10–12), and BAF-G68 (lanes 13–15).
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Figure 2: Induction of c-myc mRNA expression in BAF transformants expressing various G-CSFR–gp130 chimeric receptors. (A) FACS® analysis for the expression levels of the chimeric receptors on the BAF transformants. Cell lines tested were as follows: BAF-G277, BAF/B03 transformants bearing a chimeric receptor, the extracellular domain of the G-CSFR and the truncated gp130 including the transmembrane and cytoplasmic 133– amino acid residues, referred to as G133 (shown as BAF-G133), G133F2 with a tyrosine (Y) to phenylalanine (F) mutation at the second tyrosine, Y759 (BAF-G133F2), G-133F3, with a Y to F mutation at the third tyrosine Y767 (BAF-G133F3), and G-68, bearing the 68 membrane-proximal amino acid residues of the cytoplasmic region of gp130 (BAF-G68). (B) Northern blot analysis for c-myc mRNA expression in BAF transformants expressing various G-CSFR–gp130 chimeric receptors. Total RNAs extracted from the various BAF transformants which had been deprived of IL-3 for 12 h and stimulated with nothing or G-CSF at 100 ng/ml for the indicated periods of time were subjected to Northern blot analysis for c-myc mRNA expression. Cell lines tested were as follows: BAF-G277 (lanes 1–3), BAF-G133 (lanes 4–6), BAF-G133F2 (lanes 7–9), G-133F3 (lanes 10–12), and BAF-G68 (lanes 13–15).

Mentions: To investigate which signals were responsible for the c-myc gene activation by the IL-6 cytokine family, we determined which region of gp130 was required for full c-myc mRNA induction. To do this, we selected BAF transformants with similar expression levels of the chimeric receptors for BAF-G277, BAF-G133, BAF-G133F2, BAF-G133F3, and BAF-G68 (Fig. 2 A). The constructs of the chimeric receptors are described in the legend to Fig. 2. Previously, we showed that the membrane-proximal region of gp130, consisting of 133–amino acid residues, is necessary and sufficient for ligand-induced cell growth (21). This region of gp130 contains two tyrosines, Y759 and Y767, which are required for activation of the SHP-2/RAS/MAPK pathway and STAT3, respectively (21, 72). In BAF-G133 cells, G-CSF induced c-myc mRNA expression at a level somewhat less than that in BAF-G277, whereas BAF-G68 showed c-myc mRNA expression at a much lower level (Fig. 2 B). The low-level c-myc mRNA expression in the BAF-G68 clone was repeatedly observed and estimated as one-seventh and one-fifth of those of BAF-G277 and BAF-G133, respectively. These results suggested that although a poorly characterized and weak signal to the c-myc gene was generated from the membrane-proximal region between 1 and 68, the region between 68 and 133 is necessary for the full activation of the c-myc gene. The use of the other transformants, BAF-G133F2 and BAF-G133F3, which express the truncated chimeric receptors with a tyrosine to phenylalanine mutation at the second (Y759) and third (Y767) tyrosine residues in the cytoplasmic domain, respectively, indicated that the third tyrosine residue, Y767, in G133 was required for the full activation of the c-myc gene (Fig. 2 B). Since this requirement is the same as for STAT3 activation (19, 72), it seemed likely that STAT3 is involved in the full c-myc induction. To test this directly, we used two other BAF transformants expressing both truncated chimeric receptors, G133, and one of the two dominant-negative STAT3 mutants (20), BAF-G133 STAT3F and BAF-G133 STAT3D. Expression of either dominant-negative STAT3 almost completely inhibited the gp130-mediated expression of both c-myc and junB mRNA (Fig. 3), the latter of which has been known to be one of IL-6–inducible immediate early response genes (66, 70, 73). The three cell lines, BAF-G133, BAF-G133 STAT3F, and BAF-G133 STAT3D, could respond to IL-3 by expressing c-myc mRNA with levels similar in each cell line but much higher than that of gp130-induced c-myc mRNA, indicating that the poor gp130-mediated induction of c-myc mRNA seen in the dominant-negative STAT3–expressing cell lines was due to the specific effect of the exogenous STAT3F and STAT3D. These results indicated a critical role for STAT3 in the gp130-mediated c-myc and junB gene activation. Considering that the c-myc mRNA induction through gp130 signals occurred without requiring de novo protein synthesis (Fig. 1 C) and that the STAT3 transcription factor was involved, it seemed likely that STAT3 directly activated transcription of the c-myc gene by binding to a cis-regulatory region(s) of the gene.


STAT3 is required for the gp130-mediated full activation of the c-myc gene.

Kiuchi N, Nakajima K, Ichiba M, Fukada T, Narimatsu M, Mizuno K, Hibi M, Hirano T - J. Exp. Med. (1999)

Induction of c-myc  mRNA expression in BAF transformants expressing various  G-CSFR–gp130 chimeric receptors. (A) FACS® analysis for the  expression levels of the chimeric  receptors on the BAF transformants. Cell lines tested were as follows: BAF-G277, BAF/B03 transformants  bearing a chimeric receptor, the extracellular domain of the G-CSFR and  the truncated gp130 including the transmembrane and cytoplasmic 133– amino acid residues, referred to as G133 (shown as BAF-G133), G133F2  with a tyrosine (Y) to phenylalanine (F) mutation at the second tyrosine,  Y759 (BAF-G133F2), G-133F3, with a Y to F mutation at the third  tyrosine Y767 (BAF-G133F3), and G-68, bearing the 68 membrane-proximal amino acid residues of the cytoplasmic region of gp130 (BAF-G68). (B) Northern blot analysis for c-myc mRNA expression in BAF  transformants expressing various G-CSFR–gp130 chimeric receptors.  Total RNAs extracted from the various BAF transformants which had  been deprived of IL-3 for 12 h and stimulated with nothing or G-CSF at  100 ng/ml for the indicated periods of time were subjected to Northern  blot analysis for c-myc mRNA expression. Cell lines tested were as follows: BAF-G277 (lanes 1–3), BAF-G133 (lanes 4–6), BAF-G133F2 (lanes  7–9), G-133F3 (lanes 10–12), and BAF-G68 (lanes 13–15).
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Related In: Results  -  Collection

Show All Figures
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Figure 2: Induction of c-myc mRNA expression in BAF transformants expressing various G-CSFR–gp130 chimeric receptors. (A) FACS® analysis for the expression levels of the chimeric receptors on the BAF transformants. Cell lines tested were as follows: BAF-G277, BAF/B03 transformants bearing a chimeric receptor, the extracellular domain of the G-CSFR and the truncated gp130 including the transmembrane and cytoplasmic 133– amino acid residues, referred to as G133 (shown as BAF-G133), G133F2 with a tyrosine (Y) to phenylalanine (F) mutation at the second tyrosine, Y759 (BAF-G133F2), G-133F3, with a Y to F mutation at the third tyrosine Y767 (BAF-G133F3), and G-68, bearing the 68 membrane-proximal amino acid residues of the cytoplasmic region of gp130 (BAF-G68). (B) Northern blot analysis for c-myc mRNA expression in BAF transformants expressing various G-CSFR–gp130 chimeric receptors. Total RNAs extracted from the various BAF transformants which had been deprived of IL-3 for 12 h and stimulated with nothing or G-CSF at 100 ng/ml for the indicated periods of time were subjected to Northern blot analysis for c-myc mRNA expression. Cell lines tested were as follows: BAF-G277 (lanes 1–3), BAF-G133 (lanes 4–6), BAF-G133F2 (lanes 7–9), G-133F3 (lanes 10–12), and BAF-G68 (lanes 13–15).
Mentions: To investigate which signals were responsible for the c-myc gene activation by the IL-6 cytokine family, we determined which region of gp130 was required for full c-myc mRNA induction. To do this, we selected BAF transformants with similar expression levels of the chimeric receptors for BAF-G277, BAF-G133, BAF-G133F2, BAF-G133F3, and BAF-G68 (Fig. 2 A). The constructs of the chimeric receptors are described in the legend to Fig. 2. Previously, we showed that the membrane-proximal region of gp130, consisting of 133–amino acid residues, is necessary and sufficient for ligand-induced cell growth (21). This region of gp130 contains two tyrosines, Y759 and Y767, which are required for activation of the SHP-2/RAS/MAPK pathway and STAT3, respectively (21, 72). In BAF-G133 cells, G-CSF induced c-myc mRNA expression at a level somewhat less than that in BAF-G277, whereas BAF-G68 showed c-myc mRNA expression at a much lower level (Fig. 2 B). The low-level c-myc mRNA expression in the BAF-G68 clone was repeatedly observed and estimated as one-seventh and one-fifth of those of BAF-G277 and BAF-G133, respectively. These results suggested that although a poorly characterized and weak signal to the c-myc gene was generated from the membrane-proximal region between 1 and 68, the region between 68 and 133 is necessary for the full activation of the c-myc gene. The use of the other transformants, BAF-G133F2 and BAF-G133F3, which express the truncated chimeric receptors with a tyrosine to phenylalanine mutation at the second (Y759) and third (Y767) tyrosine residues in the cytoplasmic domain, respectively, indicated that the third tyrosine residue, Y767, in G133 was required for the full activation of the c-myc gene (Fig. 2 B). Since this requirement is the same as for STAT3 activation (19, 72), it seemed likely that STAT3 is involved in the full c-myc induction. To test this directly, we used two other BAF transformants expressing both truncated chimeric receptors, G133, and one of the two dominant-negative STAT3 mutants (20), BAF-G133 STAT3F and BAF-G133 STAT3D. Expression of either dominant-negative STAT3 almost completely inhibited the gp130-mediated expression of both c-myc and junB mRNA (Fig. 3), the latter of which has been known to be one of IL-6–inducible immediate early response genes (66, 70, 73). The three cell lines, BAF-G133, BAF-G133 STAT3F, and BAF-G133 STAT3D, could respond to IL-3 by expressing c-myc mRNA with levels similar in each cell line but much higher than that of gp130-induced c-myc mRNA, indicating that the poor gp130-mediated induction of c-myc mRNA seen in the dominant-negative STAT3–expressing cell lines was due to the specific effect of the exogenous STAT3F and STAT3D. These results indicated a critical role for STAT3 in the gp130-mediated c-myc and junB gene activation. Considering that the c-myc mRNA induction through gp130 signals occurred without requiring de novo protein synthesis (Fig. 1 C) and that the STAT3 transcription factor was involved, it seemed likely that STAT3 directly activated transcription of the c-myc gene by binding to a cis-regulatory region(s) of the gene.

Bottom Line: STAT3 does so most likely by binding to cis-regulatory region(s) of the c-myc gene.We show that STAT3 binds to a region overlapping with the E2F site in the c-myc promoter and this site is critical for the c-myc gene promoter- driven transcriptional activation by IL-6 or gp130 signals.This is the first identification of the linkage between a member of the STAT family and the c-myc gene activation, and also explains how the IL-6 family of cytokines is capable of inducing the expression of the c-myc gene.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Oncology, Biomedical Research Center, Osaka University Medical School, Suita, Osaka 565-0871, Japan.

ABSTRACT
The signal transducers and activators of transcription (STAT) family members have been implicated in regulating the growth, differentiation, and death of normal and transformed cells in response to either extracellular stimuli, including cytokines and growth factors, or intracellular tyrosine kinases. c-myc expression is coordinately regulated by multiple signals in these diverse cellular responses. We show that STAT3 mostly mediates the rapid activation of the c-myc gene upon stimulation of the interleukin (IL)-6 receptor or gp130, a signal transducing subunit of the receptor complexes for the IL-6 cytokine family. STAT3 does so most likely by binding to cis-regulatory region(s) of the c-myc gene. We show that STAT3 binds to a region overlapping with the E2F site in the c-myc promoter and this site is critical for the c-myc gene promoter- driven transcriptional activation by IL-6 or gp130 signals. This is the first identification of the linkage between a member of the STAT family and the c-myc gene activation, and also explains how the IL-6 family of cytokines is capable of inducing the expression of the c-myc gene.

Show MeSH
Related in: MedlinePlus