Article Figures & Data
Figures
- Fig. 1.
Induction of C/EBPδ and C/EBPβ mRNAs by IL-6 or IFN-γ in hepatoma cell lines. (A) Northern blot analysis of 20 μg of total RNA from Hep3B or HepG2 cells cells treated with IL-6 or IFN-γ for the indicated times. Duplicate blots were hybridized with C/EBPδ or C/EBPβ probes and then with cyclophilin. (B) Northern blotting analysis of 20 μg of total RNAs from control Hep3B cells (lane 1), cells treated with cycloheximide (CHX) for 30, 60, and 120 min (lanes 2 to 4), and cells pretreated with cycloheximide for 30, 60, and 120 min followed by the addition of IL-6 for 2 h (lanes 5 to 7), IL-6 alone for 2 h (lane 8), or cycloheximide and IL-6 concurrently for 2 h (lane 9). The blots were hybridized sequentially with the indicated probes.
- Fig. 2.
DNA sequence of the murine C/EBPδ promoter and identification of several putative regulatory elements. The sequence extends from a ScaI site at −729 to position +12 relative to the transcription startsite. Endpoints of 5′ deletion mutants are indicated by brackets at −729, −322, −127, −81, and −36. Sequences corresponding to potential regulatory sites are boxed. The arrow denotes the transcription startsite (25).
- Fig. 3.
Identification of an IL-6-responsive region within the C/EBPδ promoter. Luciferase reporter constructs containing the indicated 5′ promoter deletions were cotransfected with pRSV β-gal into Hep3B cells. The luciferase data were normalized to β-galactosidase values to control for differences in transfection efficiencies. The values represent averages ± standard deviations of three independent experiments. Basal luciferase expression levels from each construct are shown relative to the value for (−127)-Luc. Basal expression from (−36)-Luc was detectable but was rounded to 0.0. Fold induction represents luciferase activity after IL-6 treatment relative to the basal level.
- Fig. 4.
Analysis of APRE and Sp1 sites. (A) Diagram of mutations. The mutations were introduced into the APRE at −106 bp and the Sp1 sites at −117 and −53 bp. The wild-type sequences are indicated at the top, and the mutated sequences are shown below. The mutations were incorporated into the (−127)-Luc deletion construct. (B) Transient transfection assays of promoter mutants. The indicated promoter-reporter constructs were cotransfected with pRSV β-gal into Hep3B cells. The luciferase data were normalized to β-galactosidase activity. The values represent the averages of three to six independent experiments. The relative basal expression and fold induction values were determined as described in the legend to Fig. 3.
- Fig. 5.
The C/EBPδ APRE competes for binding of Stat3 to the α2-m APRE. (A) EMSA using the rat α2-m APRE, nuclear extracts (6.5 μg) from HepG2 cells, and NRS or Stat3-specific antibody (Ab) as indicated. The HepG2 cells were treated with IL-6 for 15 min. An upper complex (u) and a lower complex (l) appear in the IL-6-treated extracts (lane 3). The Stat3 antibody supershift complex (lane 4) is indicated. (B) Competition for Stat3 binding by the C/EBPδ APRE. Nuclear extracts (10 μg) from IL-6 treated HepG2 cells were incubated with Stat3-specific antibody and 10× (lanes 3 and 6), 30× (lanes 4 and 7), or 100× (lanes 5 and 8) molar excess of unlabeled wild-type (δAPRE) or mutant (δAPREm) binding site, as indicated. The rat α2-m APRE was used as a probe. The film was overexposed to emphasize the supershifted complex.
- Fig. 6.
Selective binding of Stat3 to the C/EBPδ APRE. Nuclear extracts from control or IL-6-treated HepG2 cells were analyzed by EMSA using the wild-type or mutant C/EBPδ APRE probes and control antiserum or Stat1- or Stat3-specific antibody (Ab), as indicated. The film was overexposed to emphasize the supershift signal.
- Fig. 7.
Binding of nuclear proteins to the Sp1(−117) site. (A) Supershift analysis using an antibody (Ab) against Sp1. Nuclear extracts were prepared from control or IL-6-treated HepG2 cells by a method that maximizes the extraction of Sp1 (see Materials and Methods). The extracts were incubated with either NRS or an Sp1-specific antibody and the δAPRE/Sp1 or δAPRE probe. The supershift generated by the Sp1 antibody in lanes 2 and 4 is indicated. (B and C) Binding of recombinant Sp1 to the δAPRE/Sp1 (B) and δAPRE (C) probes. Recombinant human Sp1 (50 ng) was used alone (lanes 5 and 10) or mixed with 8 μg of HepG2 nuclear extract (optimized for Stat protein extraction) from control or IL-6-treated cells. Stat3 antibody was added to the indicated reactions. The lower panel is a longer exposure of the top portion of the gel to emphasize the Stat3 antibody supershift complex.
- Fig. 8.
The C/EBPδ APRE confers IL-6 responsiveness to a heterologous promoter. One or four copies of the C/EBPδ APRE or six copies of the α2-m APRE were inserted upstream of the TK promoter. The constructs were tested for induction by IL-6 after transfection into Hep3B cells as described in the legend to Fig. 3.
- Fig. 9.
Stat3 mediates IL-6-induced expression from the C/EBPδ promoter. (A) Stat3 but not Stat1 transactivates the C/EBPδ promoter. The (−127)-Luc construct was cotransfected into Hep3B cells with expression vectors for Stat1 or Stat3 or the parental pCDNA1 vector, together with pRSV β-gal as an internal standard, and tested for basal and IL-6-induced luciferase expression. (B) Stat3 transactivation of C/EBPδ promoter mutants. The indicated deletion and point mutants (Fig. 3 and 4) were cotransfected with the Stat3 expression vector into Hep3B cells and tested for basal and IL-6-inducible luciferase expression. (C) Stat3 transactivates a heterologous promoter containing the C/EBPδ APRE. The indicated TK promoter-luciferase reporter constructs (Fig. 8) were cotransfected with the Stat3 expression plasmid into Hep3B cells and tested for basal and IL-6-inducible expression. The cell extracts were assayed for luciferase and β-galactosidase activities as described in Fig. 3. The values represent the averages of three to six independent experiments.
- Fig. 10.
Replacement of the C/EBPδ APRE with SBEs from ICAM-1 or C/EBPβ renders the promoter responsive to IFN-γ. (A) The C/EBPβ promoter contains an SBE that binds Stat1 and Stat3. A probe containing the putative SBE from the C/EBPβ promoter and Stat1- or α-Stat3-specific antibody (Ab) (lanes 3 and 4, respectively) were added to nuclear extracts from control (lane 1) or IL-6 treated (lanes 2 to 4) HepG2 cells and the reactions analyzed by EMSA. Antibody supershift species are indicated. (B) Comparison of SBE sequences from the C/EBPδ, C/EBPβ, and ICAM-1 promoters. Bases that differ from the C/EBPδ APRE sequence are underlined. (C) IL-6 and IFN-γ responsiveness of SBE swap mutants. Constructs in which the C/EBPδ APRE was exchanged with SBEs from the C/EBPβ or ICAM-1 genes were generated. These constructs and (−127)-Luc were cotransfected with pRSV β-gal into Hep3B cells and assayed for basal expression and IL-6 or IFN-γ inducibility. The values represent the average of three independent experiments. Relative basal expression was normalized to the (−127)-Luc level.
Tables
- Table 1.
Sequences and Stat binding properties of the C/EBPδ APRE and several known SBEs
Gene SBEa Binding to: Reference(s) Stat1 Stat3 Murine C/EBPδ TCG TT CCC AGCAGCA − + This study Rat C/EBPβ TGT TT CCC AGAAGTT + + This study Rat α2-m GAATT CCC AGAA GGA + + 17, 27 Human hemopexin TTT TT CCC GGCA GGA ND ND 22 Rat α1-acid glycoprotein TTT TT CCC AGAA GCC ND ND 49 Human haptoglobin CTT TT CCA GTAACAA ND ND 32 Human γ-fibrinogen TTC TT CCA GTAC ATG ND + 51 Human α1-antichymotrypsin CTA TT ACA G A AAA TT ND ND 4 Human C-reactive protein CTC TT CCC G A AGCTC ND + 50 Human LBP AGATT CCC AGTGCAG ND + 41 Murine junB CGCTT CCT G A CAGTG − + 12 Human c-fos CAGTT CCC GTCAA TC + + 8 Human ICAM-1 GCT TT CCC GGAAA CC + + 7, 28 ↵a SBEs from a number of cytokine-inducible genes are shown in alignment with the C/EBPδ APRE. The sequences were aligned through the core palindromic TT-AA motif (boldface). In cases where there is more than one possibility for the -AA motif, the alternative bases are boldface and underlined.
b Binding (+) or lack of binding (−) of each SBE to Stat1 and Stat3, as determined by use of specific antibodies in supershift assays. ND, not determined.
