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. 2007 Mar 15;402(3):567-74.
doi: 10.1042/BJ20061549.

Transcriptional repression of the gluconeogenic gene PEPCK by the orphan nuclear receptor SHP through inhibitory interaction with C/EBPalpha

Min Jung Park  1 , Hee Jeong Kong, Hye Young Kim, Hyeong Hoe Kim, Joon Hong Kim, Jae Hun Cheong
Affiliations

Transcriptional repression of the gluconeogenic gene PEPCK by the orphan nuclear receptor SHP through inhibitory interaction with C/EBPalpha

Min Jung Park et al. Biochem J. .

Abstract

SHP (short heterodimer partner) is an orphan nuclear receptor that plays an important role in regulating glucose and lipid metabolism. A variety of transcription factors are known to regulate transcription of the PEPCK (phosphoenolpyruvate carboxykinase) gene, which encodes a rate-determining enzyme in hepatic gluconeogenesis. Previous reports identified glucocorticoid receptor and Foxo1 as novel downstream _targets regulating SHP inhibition [Borgius, Steffensen, Gustafsson and Treuter (2002) J. Biol. Chem. 277, 49761-49796; Yamagata, Daitoku, Shimamoto, Matsuzaki, Hirota, Ishida and Fukamizu (2004) J. Biol. Chem. 279, 23158-23165]. In the present paper, we show a new molecular mechanism of SHP-mediated inhibition of PEPCK transcription. We also show that the CRE1 (cAMP regulatory element 1; -99 to -76 bp relative to the transcription start site) of the PEPCK promoter is also required for the inhibitory regulation by SHP. SHP repressed C/EBPalpha (CCAAT/enhancer-binding protein alpha)-driven transcription of PEPCK through direct interaction with C/EBPalpha protein both in vitro and in vivo. The formation of an active transcriptional complex of C/EBPalpha and its binding to DNA was inhibited by SHP, resulting in the inhibition of PEPCK gene transcription. Taken together, these results suggest that SHP might regulate a level of hepatic gluconeogenesis driven by C/EBPalpha activation.

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Figures

Figure 1
Figure 1. SHP represses transcriptional activation of the PEPCK promoter
(A) Schematic representation of two luciferase reporter constructs of the PEPCK promoter are shown. Region A contains the glucocorticoid response unit composed of two glucocorticoid regulatory elements (GR1 and 2), three accessory factor-binding sites (AF1–3) and a CRE. This region provides several factor-binding sites for RAR, RXR, GR, TR, C/EBP, and HNF-3 [,–17]. Region B contains CRE1 (at −99 to −76 bp relative to the transcription start site) and is immediately adjacent to a nuclear factor 1-binding site (NF1). (B) SHP decreases the transcriptional activity of the PEPCK promoter. HepG2 cells were transiently transfected with reporter constructs PEPCK-275 or PEPCK-543 along with the indicated expression plasmids. The cells were incubated for 48 h after transfection, then harvested and the luciferase activity measured. All the transfection results were normalized to β-galactosidase activity, and the results represent the means±S.E.M. for three independent experiments, with fold induction over the level observed with the reporter alone.
Figure 2
Figure 2. SHP inhibits C/EBPα-mediated transcription activation of the PEPCK promoter
(A) An expression vector for C/EBPα and increasing amounts of SHP were co-transfected into HepG2 cells with the reporter construct PEPCK-CRE1. The expression level of C/EBPα (HA-tagged) was confirmed by Western blotting using an antibody against the HA-tag. (B) The inhibitory function of SHP on C/EBPα transcription activation is dependent on the CRE1 sequence of the PEPCK promoter. The same transient transfection assay as shown in (A) was performed using wild-type PEPCK-275-luc and mutant PEPCK-275m-luc reporter constructs instead of PEPCK-CRE1. The mutated sequence of the CRE1 of PEPCK-275m-luc was described in the Experimental section. The cells were incubated for 48 h after transfection, then harvested and the luciferase activity measured. All the transfection results were normalized to β-galactosidase activity, and the results represent the means±S.E.M for four independent experiments, with fold induction over the level observed with the reporter alone.
Figure 3
Figure 3. SHP interacts with C/EBPα
(A) SHP co-precipitates with C/EBPα from rat liver nuclear extracts. SHP and C/EBPα were co-immunoprecipitated (IP) from rat liver nuclear extract by incubation with anti-C/EBPα IgG. Antibody complexes were captured on protein A–Sepharose. The beads were washed three times with binding buffer and eluted into Laemmli sample buffer. Proteins were resolved by SDS/PAGE (10% gels), electroblotted and detected by Western blotting by using an anti-SHP antibody. Lane 1, rat liver nuclear extract; lane 2, co-immunoprecipitates from samples using preimmune serum; lane 3, co-immunoprecipitates from samples treated with anti-C/EBPα IgG. (B) The N-terminal domain of C/EBPα interacts with SHP in vitro. GST fusions to full-length (C/EBPα-F), N-terminal domain (C/EBPα-N) and C-terminal domain (C/EBPα-C) of C/EBPα were purified from E. coli. In vitro translated SHP protein in the presence of [35S]-methionine were incubated with GST or GST-fusion proteins immobilized on glutathione–resin. The bound proteins were eluted by GSH and resolved by SDS/PAGE. (C) SHP associates with the N-terminal transcriptional activation domain of C/EBPα in cells. The mammalian expression plasmids encoding VP16–SHP, and GAL4–full length C/EBPα (F), Gal4–N-terminal (N) and Gal4–C-terminal (C) domains were transfected into HepG2 cells as indicated. The cells were incubated for 48 h after transfection, then harvested and the luciferase activity measured. The results represent the means for three independent experiments, with fold induction over the level observed with the reporter alone.
Figure 4
Figure 4. Interaction region of SHP with C/EBPα in yeast two-hybrid assay
An illustration of the domain structure and deletion constructs of SHP (A) and C/EBPα (B). INT, interaction domain of SHP; REP, repression domain of SHP; TAD, transcriptional activation and regulatory domain of C/EBPα; bZIP, basic and leucine zipper domain of C/EBPα. (C) The indicated B42 and LexA plasmids were transformed into EGY48 yeast cells containing the appropriate β-galactosidase reporter gene. At least three separate transformants from each transformation were transferred to indicator plates containing 5-bromo-4-chloroindol-3-yl β-D-galactopyranoside (X-Gal) and reproducible results were obtained using colonies from a separate transformation. The number indicates the β-galactosidase activity, measured using the β-galactosidase enzyme assay (Promega).
Figure 5
Figure 5. SHP inhibits the DNA binding and homo/heterodimerization of C/EBPα
(A) SHP blocks C/EBPα–DNA complex formation. A double-stranded oligonucleotide probe containing the CRE1 site of the PEPCK promoter was used in EMSAs. Rat liver nuclear extracts (20 ng) were incubated as indicated, followed by the addition of the probe. Mobility shifts were demonstrated by incubation of the nuclear extract proteins with 100 ng of GST–RXR or SHP proteins. The specificty of the C/EBPα–CRE complex was confirmed by incubating the nuclear extracts with anti-C/EBPα IgG. (B) Bacterially expressed and purified GST–C/EBPα, GST–RXR and GST–SHP were incubated as indicated, followed by the addition of a DNA probe. Mobility shifts were demonstrated by incubation of 50 ng of GST–C/EBPα with increasing amounts of GST–RXR or SHP proteins (50 and 100 ng). (C) SHP inhibits the homo- and hetero-dimerization of C/EBPα. The mammalian expression plasmids encoding VP16–C/EBPα or –ATF-2 and GAL4–C/EBPα were transiently transfected with the Gal4-tk-luc reporter plasmid into HepG2 cells as indicated. The cells were incubated for 48 h after transfection, then harvested and the luciferase activity measured. The results represent the means for three independent experiments, with fold induction over the level observed with the reporter alone. C, C/EBPα; A, ATF-2.
Figure 6
Figure 6. SHP inhibits the functional complex of C/EBPα and RFC140
(A) SHP inhibits the co-activator function of RFC140 in C/EBPα-mediated transcription activation. Cells were co-transfected with constructs encoding C/EBPα and RFC140 and increasing amounts of SHP, along with the PEPCK-275-luc promoter–reporter construct. Results are the means for three independent experiments. (B) SHP inhibits the protein–protein interaction between C/EBPα and RFC140. GST fusions to the C/EBPα-interaction region (residues 151–545) of RFC140 were purified from E. coli. C/EBPα was in vitro translated in the presence of 35S-methionine and then incubated with glutathione-resin immobilized GST–RFC140(151–545) in the presence of increasing amounts of GST–SHP or GST alone. The bound proteins were eluted by GSH and resolved by SDS/PAGE.
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