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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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The nature of the gp130-signal–induced DNA binding activities at the  c-myc promoter E2F site. Nuclear extracts  from (A) IL-3–deprived BAF-G277 cells  and (B) serum-starved HepG2 cells either  untreated (A and B, lane 1) or treated with  G-CSF (A, lanes 2–5) or IL-6 (B, lanes 2–4)  at 100 ng/ml for 15 min were preincubated  without (A and B, lanes 1 and 2) or with  anti-STAT1 mAb (A and B, lane 3), anti-STAT3 antibody (A and B, lane 4), or anti-STAT5 antibody (A, lane 5) for 30 min on  ice before the addition of 32P-labeled c-myc  E2F oligonucleotides, and were then subjected to electrophoresis and autoradiography. The positions of the gp130-signal  inducible complexes containing STAT3,  STAT3/STAT1, and STAT1 are indicated.  (C) Nuclear extracts from serum-starved  HepG2 either unstimulated (lanes 1 and 3) or stimulated with IL-6 for 15 min (lanes 2 and 4) were subjected to EMSA with c-myc E2F oligonucleotide  probe (lanes 1 and 2) and APRE oligonucleotide probe (lanes 3 and 4). (D) Binding specificity of IL-6–inducible c-myc E2F site–binding complexes. Nuclear extracts from serum-starved HepG2 cells either untreated (lane 1) or treated with IL-6 (lanes 2–8) were preincubated with unlabeled oligonucleotide competitors (50- or 250-fold molar excess as indicated) for 5 min, followed by incubation with labeled c-myc E2F probes. The competitors used  were unlabeled oligonucleotides containing the c-myc E2F site (lanes 3 and 4), the adenovirus E2 E2F site (lanes 5 and 6), or the α2 macroglobulin  APRE (lanes 7 and 8).
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Figure 7: The nature of the gp130-signal–induced DNA binding activities at the c-myc promoter E2F site. Nuclear extracts from (A) IL-3–deprived BAF-G277 cells and (B) serum-starved HepG2 cells either untreated (A and B, lane 1) or treated with G-CSF (A, lanes 2–5) or IL-6 (B, lanes 2–4) at 100 ng/ml for 15 min were preincubated without (A and B, lanes 1 and 2) or with anti-STAT1 mAb (A and B, lane 3), anti-STAT3 antibody (A and B, lane 4), or anti-STAT5 antibody (A, lane 5) for 30 min on ice before the addition of 32P-labeled c-myc E2F oligonucleotides, and were then subjected to electrophoresis and autoradiography. The positions of the gp130-signal inducible complexes containing STAT3, STAT3/STAT1, and STAT1 are indicated. (C) Nuclear extracts from serum-starved HepG2 either unstimulated (lanes 1 and 3) or stimulated with IL-6 for 15 min (lanes 2 and 4) were subjected to EMSA with c-myc E2F oligonucleotide probe (lanes 1 and 2) and APRE oligonucleotide probe (lanes 3 and 4). (D) Binding specificity of IL-6–inducible c-myc E2F site–binding complexes. Nuclear extracts from serum-starved HepG2 cells either untreated (lane 1) or treated with IL-6 (lanes 2–8) were preincubated with unlabeled oligonucleotide competitors (50- or 250-fold molar excess as indicated) for 5 min, followed by incubation with labeled c-myc E2F probes. The competitors used were unlabeled oligonucleotides containing the c-myc E2F site (lanes 3 and 4), the adenovirus E2 E2F site (lanes 5 and 6), or the α2 macroglobulin APRE (lanes 7 and 8).

Mentions: Since the sequence of the c-myc E2F binding site, TTGGCGGGAAA, is distinct from the known STAT3 binding sites (76–78), we tested whether STAT3 and other STAT proteins bind directly to the c-myc E2F site or indirectly through interactions with other proteins. To do this, we used EMSA, testing nuclear extracts from BAF-G277 and HepG2 cells stimulated with either G-CSF or IL-6. The extracts were incubated with labeled oligonucleotides bearing the c-myc E2F site and specific antibodies to each STAT protein in some experiments and unlabeled oligonucleotides as competitors in other experiments. The nuclear extracts from BAF-G277 cells stimulated with G-CSF for 15 min (Fig. 7 A, lanes 1–5) and HepG2 cells stimulated with IL-6 for 15 min (Fig. 7 B, lanes 1–4) have prominent c-myc E2F site binding activity. In the nuclear extracts from BAF-G277 cells stimulated with G-CSF, most of the complexes on the c-myc E2F site were shifted by anti-STAT3 serum (Fig. 7 B, lane 4), indicating that STAT3 is the major component bound to the c-myc E2F site in BAF-G277 cells, which have very little E2F activity, either free E2F or complexes of E2F and one of the retinoblastoma protein family members. The inducible complexes in the IL-6– stimulated HepG2 nuclear extracts were most likely to contain STAT3, STAT3/STAT1, and STAT1 (indicated by arrows in Fig. 7 B), since anti-STAT3 serum shifted the two upper bands and anti-STAT1 antibody shifted the lower band. Interestingly, the mobilities of the complexes with the c-myc E2F probe are exactly the same as those of complexes seen using an oligonucleotide probe containing an APRE from α2-macroglobulin, a typical STAT binding site (compare lane 2 with lane 4 in Fig. 7 C), suggesting that these inducible complexes with c-myc E2F probe are dimers of STAT3, STAT3/STAT1, and STAT1, although c-myc E2F probe has less affinity than APRE probe. These inducible complexes of STAT1 and 3 with the c-myc E2F probe were competed by an oligonucleotide containing APRE (Fig. 7 D, lanes 7 and 8) five times as efficiently as by a control c-myc E2F oligonucleotide (Fig. 7 D, lanes 3 and 4), but not by the typical E2F binding site, TTTCGCGC, taken from the adenovirus E2 promoter (Fig. 7 D, lanes 5 and 6). These results suggested that activated STAT proteins may directly bind to the c-myc E2F site with around fivefold less affinity than to APRE and that in spite of the low affinity to the c-myc E2F site, the binding activities of STAT proteins were much stronger than those of E2F-containing complexes.


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)

The nature of the gp130-signal–induced DNA binding activities at the  c-myc promoter E2F site. Nuclear extracts  from (A) IL-3–deprived BAF-G277 cells  and (B) serum-starved HepG2 cells either  untreated (A and B, lane 1) or treated with  G-CSF (A, lanes 2–5) or IL-6 (B, lanes 2–4)  at 100 ng/ml for 15 min were preincubated  without (A and B, lanes 1 and 2) or with  anti-STAT1 mAb (A and B, lane 3), anti-STAT3 antibody (A and B, lane 4), or anti-STAT5 antibody (A, lane 5) for 30 min on  ice before the addition of 32P-labeled c-myc  E2F oligonucleotides, and were then subjected to electrophoresis and autoradiography. The positions of the gp130-signal  inducible complexes containing STAT3,  STAT3/STAT1, and STAT1 are indicated.  (C) Nuclear extracts from serum-starved  HepG2 either unstimulated (lanes 1 and 3) or stimulated with IL-6 for 15 min (lanes 2 and 4) were subjected to EMSA with c-myc E2F oligonucleotide  probe (lanes 1 and 2) and APRE oligonucleotide probe (lanes 3 and 4). (D) Binding specificity of IL-6–inducible c-myc E2F site–binding complexes. Nuclear extracts from serum-starved HepG2 cells either untreated (lane 1) or treated with IL-6 (lanes 2–8) were preincubated with unlabeled oligonucleotide competitors (50- or 250-fold molar excess as indicated) for 5 min, followed by incubation with labeled c-myc E2F probes. The competitors used  were unlabeled oligonucleotides containing the c-myc E2F site (lanes 3 and 4), the adenovirus E2 E2F site (lanes 5 and 6), or the α2 macroglobulin  APRE (lanes 7 and 8).
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Figure 7: The nature of the gp130-signal–induced DNA binding activities at the c-myc promoter E2F site. Nuclear extracts from (A) IL-3–deprived BAF-G277 cells and (B) serum-starved HepG2 cells either untreated (A and B, lane 1) or treated with G-CSF (A, lanes 2–5) or IL-6 (B, lanes 2–4) at 100 ng/ml for 15 min were preincubated without (A and B, lanes 1 and 2) or with anti-STAT1 mAb (A and B, lane 3), anti-STAT3 antibody (A and B, lane 4), or anti-STAT5 antibody (A, lane 5) for 30 min on ice before the addition of 32P-labeled c-myc E2F oligonucleotides, and were then subjected to electrophoresis and autoradiography. The positions of the gp130-signal inducible complexes containing STAT3, STAT3/STAT1, and STAT1 are indicated. (C) Nuclear extracts from serum-starved HepG2 either unstimulated (lanes 1 and 3) or stimulated with IL-6 for 15 min (lanes 2 and 4) were subjected to EMSA with c-myc E2F oligonucleotide probe (lanes 1 and 2) and APRE oligonucleotide probe (lanes 3 and 4). (D) Binding specificity of IL-6–inducible c-myc E2F site–binding complexes. Nuclear extracts from serum-starved HepG2 cells either untreated (lane 1) or treated with IL-6 (lanes 2–8) were preincubated with unlabeled oligonucleotide competitors (50- or 250-fold molar excess as indicated) for 5 min, followed by incubation with labeled c-myc E2F probes. The competitors used were unlabeled oligonucleotides containing the c-myc E2F site (lanes 3 and 4), the adenovirus E2 E2F site (lanes 5 and 6), or the α2 macroglobulin APRE (lanes 7 and 8).
Mentions: Since the sequence of the c-myc E2F binding site, TTGGCGGGAAA, is distinct from the known STAT3 binding sites (76–78), we tested whether STAT3 and other STAT proteins bind directly to the c-myc E2F site or indirectly through interactions with other proteins. To do this, we used EMSA, testing nuclear extracts from BAF-G277 and HepG2 cells stimulated with either G-CSF or IL-6. The extracts were incubated with labeled oligonucleotides bearing the c-myc E2F site and specific antibodies to each STAT protein in some experiments and unlabeled oligonucleotides as competitors in other experiments. The nuclear extracts from BAF-G277 cells stimulated with G-CSF for 15 min (Fig. 7 A, lanes 1–5) and HepG2 cells stimulated with IL-6 for 15 min (Fig. 7 B, lanes 1–4) have prominent c-myc E2F site binding activity. In the nuclear extracts from BAF-G277 cells stimulated with G-CSF, most of the complexes on the c-myc E2F site were shifted by anti-STAT3 serum (Fig. 7 B, lane 4), indicating that STAT3 is the major component bound to the c-myc E2F site in BAF-G277 cells, which have very little E2F activity, either free E2F or complexes of E2F and one of the retinoblastoma protein family members. The inducible complexes in the IL-6– stimulated HepG2 nuclear extracts were most likely to contain STAT3, STAT3/STAT1, and STAT1 (indicated by arrows in Fig. 7 B), since anti-STAT3 serum shifted the two upper bands and anti-STAT1 antibody shifted the lower band. Interestingly, the mobilities of the complexes with the c-myc E2F probe are exactly the same as those of complexes seen using an oligonucleotide probe containing an APRE from α2-macroglobulin, a typical STAT binding site (compare lane 2 with lane 4 in Fig. 7 C), suggesting that these inducible complexes with c-myc E2F probe are dimers of STAT3, STAT3/STAT1, and STAT1, although c-myc E2F probe has less affinity than APRE probe. These inducible complexes of STAT1 and 3 with the c-myc E2F probe were competed by an oligonucleotide containing APRE (Fig. 7 D, lanes 7 and 8) five times as efficiently as by a control c-myc E2F oligonucleotide (Fig. 7 D, lanes 3 and 4), but not by the typical E2F binding site, TTTCGCGC, taken from the adenovirus E2 promoter (Fig. 7 D, lanes 5 and 6). These results suggested that activated STAT proteins may directly bind to the c-myc E2F site with around fivefold less affinity than to APRE and that in spite of the low affinity to the c-myc E2F site, the binding activities of STAT proteins were much stronger than those of E2F-containing complexes.

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