Regulation of c-myc expression by IFN-gamma through Stat1-dependent and -independent pathways

Abstract

Interferons (IFNs) inhibit cell growth in a Stat1-dependent fashion that involves regulation of c-myc expression. IFN-gamma suppresses c-myc in wild-type mouse embryo fibroblasts, but not in Stat1-null cells, where IFNs induce c-myc mRNA rapidly and transiently, thus revealing a novel signaling pathway. Both tyrosine and serine phosphorylation of Stat1 are required for suppression. Induced expression of c-myc is likely to contribute to the proliferation of Stat1-null cells in response to IFNs. IFNs also suppress platelet-derived growth factor (PDGF)-induced c-myc expression in wild-type but not in Stat1-null cells. A gamma-activated sequence element in the promoter is necessary but not sufficient to suppress c-myc expression in wild-type cells. In PKR-null cells, the phosphorylation of Stat1 on Ser727 and transactivation are both defective, and c-myc mRNA is induced, not suppressed, in response to IFN-gamma. A role for Raf-1 in the Stat1-independent pathway is revealed by studies with geldanamycin, an HSP90-specific inhibitor, and by expression of a mutant of p50(cdc37) that is unable to recruit HSP90 to the Raf-1 complex. Both agents abrogated the IFN-gamma-dependent induction of c-myc expression in Stat1-null cells.

Fig. 1. c–myc mRNA expression in response to IFN–γ in Stat1-null and wild-type MEFs. (A) Subconfluent, serum-starved MEFs were either untreated or treated with 1000 IU/ml of murine IFN–γ for 15 or 30 min. c–myc and GAPDH mRNA levels were analyzed by Northern blotting. (B) Stat1-null cells were treated with murine IFN–γ (1000 IU/ml). c–myc and GAPDH mRNA levels were determined as above. (C) Wild-type cells were treated with murine IFN–γ (1000 IU/ml). c–myc and GAPDH mRNA levels were determined as above. (D) Subconfluent, serum-starved fibroblasts were either untreated or treated with 1000 IU/ml of murine IFN–γ for 30 min. Northern blot analyses were conducted with the probes indicated.

Fig. 2. Effects of growth factor and IFN treatment on c–myc regulation. (A) MEFs were grown to 20% confluence in DMEM with 10% FCS. The cells were serum-starved in DMEM with 0.1% FCS for 48 h. Cells were either untreated (–) or treated with 10% FCS, alone (FCS) or with 1000 IU/ml of murine IFN–γ (FCS+IFN–γ). Twenty-four hours later, the cells were stained with propidium iodide and the DNA content was analyzed by flow cytometry. The percentage of cells in the G₁, S and G₂+M parts of the cell cycle are indicated in each histogram. (B) Stat1-null or wild-type MEFs were either untreated or treated for 30 min with 1000 IU/ml of IFN–γ alone, 200 ng/ml of PDGF alone, or PDGF plus IFN–γ. Northern transfers were hybridized with c–myc or GAPDH probes. (C) Stat1-null or wild-type MEFs were either untreated or treated for 30 min with 1000 IU/ml of IFN–β alone, 200 ng/ml of PDGF alone, or PDGF plus IFN–β. Northern transfers were hybridized with c–myc or GAPDH probes.

Fig. 3. Regulation of c–myc but not p21^waf1 expression by IFN–γ in 2fTGH and U3A cells. (A) Subconfluent, serum-starved cells were treated with 1500 IU/ml of human IFN–γ for 60 to 120 min. Northern transfers were hybridized with the probes indicated. (B) Cells were either untreated or treated with 1500 IU/ml of human IFN–γ for 3 or 6 h. p21^waf1 levels were determined by Western blot analysis.

Fig. 4. Stat1 domains required to suppress expression of c–myc mRNA. U3A cells reconstituted with a Stat1 tyrosine (U3A Stat1 Y701F) or serine (U3A Stat1 S727A) phosphorylation site mutant or with wild-type Stat1α (Kumar et al., 1997b; see for expression levels) were treated with 1500 IU/ml of human IFN–γ. c–myc and GAPDH RNA levels were determined by Northern blot analysis.

Fig. 5. Identification of a GAS element in the c–myc promoter that is necessary but not sufficient for suppression. (A) The full-length 1.7 kb c–myc promoter or two deletion constructs were linked to luciferase and transfected transiently into 2fTGH cells. Luciferase activity was determined with or without IFN–γ treatment (1500 IU/ml for 6 h). The data shown represent duplicate experiments in three independent trials (standard deviations). (B) Constructs containing eight copies of the consensus GAS (TTCTCGGAA) or seven copies of the c–myc GAS (TTCTGGGAA) linked to luciferase were transfected transiently into 2fTGH cells. Luciferase activity was determined with or without IFN–γ treatment (1500 IU/ml for 6 h). Data are presented from three independent experiments, with standard deviations. (C) NIH 3T3 cells stably transfected with the 1.7 kb c–myc promoter linked to the cell-surface protein cd2 were serum-starved and treated with PDGF alone (200 ng/ml), PDGF plus IFN–γ (1000 IU/ml) or were untreated. cd2 expression was determined by FACScan analysis.

Fig. 6. Binding of Stat1 to the c–myc GAS. (A) EMSAs were performed with whole-cell extracts prepared from U3A or 2fTGH cells, not treated or treated for 15 or 30 min with 1000 IU/ml of IFN–γ. (B) EMSAs were performed with whole-cell extracts from 2fTGH cells treated with 1000 IU/ml of IFN–γ. The extracts were pre-incubated with anti-Stat1 or with a 100–fold molar excess of the unlabeled SIE (m67) GAS. (C) EMSAs were performed with whole-cell extracts from NIH 3T3 cells, treated for 30 min with 200 ng/ml of PDGF, 1000 IU/ml of murine IFN–γ, or not treated.

Fig. 7. Defective Stat1 activation and serine phosphorylation in PKR-null cells. (A) 8× con GAS linked to luciferase was transiently transfected into wild-type or PKR-null MEFs. Luciferase activity was determined with or without murine IFN–γ (1000 IU/ml) for 6 h. Results are presented with standard deviations from three independent experiments. (B) Whole-cell extracts were prepared from serum-starved wild-type or PKR-null cells, with or without treatment with 1000 IU/ml of murine IFN–γ. Stat1 binding to the SIE (m67) GAS was determined by EMSA. (C) Extracts of serum-starved cells, either untreated or treated with IFN–γ (1000 IU/ml for 20 min) were immunoprecipitated with anti-Stat1. The transfer was probed first with an antibody specific for a Stat1 peptide that includes phosphorylated Ser727 and then reprobed with anti-Stat1. (D) RNA from MEFs untreated or treated with murine IFN–γ (1000 IU/ml) was analyzed by the Northern procedure.

Fig. 7. Defective Stat1 activation and serine phosphorylation in PKR-null cells. (A) 8× con GAS linked to luciferase was transiently transfected into wild-type or PKR-null MEFs. Luciferase activity was determined with or without murine IFN–γ (1000 IU/ml) for 6 h. Results are presented with standard deviations from three independent experiments. (B) Whole-cell extracts were prepared from serum-starved wild-type or PKR-null cells, with or without treatment with 1000 IU/ml of murine IFN–γ. Stat1 binding to the SIE (m67) GAS was determined by EMSA. (C) Extracts of serum-starved cells, either untreated or treated with IFN–γ (1000 IU/ml for 20 min) were immunoprecipitated with anti-Stat1. The transfer was probed first with an antibody specific for a Stat1 peptide that includes phosphorylated Ser727 and then reprobed with anti-Stat1. (D) RNA from MEFs untreated or treated with murine IFN–γ (1000 IU/ml) was analyzed by the Northern procedure.

Fig. 8. Pre-treatment with geldanamycin or dominant-negative p50^cdc37 abrogates the induction of c–myc by IFN–γ. (A) Stat1-null MEFs were serum-starved and either untreated or pre-treated with geldanamycin (GA, 2 μg/ml) for 5 h. The cells were unstimulated or stimulated with IFN–γ, IFN–β or PDGF for 30 min. c–myc and GAPDH mRNA levels were determined by Northern blot analysis. (B) Stat1-null MEFs were transiently transfected with vector alone or vector encoding dominant-negative p50^cdc37. The transfected cells were serum-starved for 24 h and either unstimulated or stimulated with IFN–γ for 30 min. c–myc and GAPDH RNA levels were determined as above.