Interleukin-6-Specific Activation of the C/EBPδ Gene in Hepatocytes Is Mediated by Stat3 and Sp1

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).

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.

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.

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 (δAPRE_m) binding site, as indicated. The rat α₂-m APRE was used as a probe. The film was overexposed to emphasize the supershifted complex.

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.

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.

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.

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.