Figure 1.
(A,B), AML12 cells were infected with Ad-GFP or Ad-ERRγ for 24 hr. (A),Total RNA was isolated for qRT-PCR analyses to quantify CREBH mRNA levels using CREBH primers. (B), Western blot analyses shows CREBH full length (CREBH-F), and CREBH active form (CREBH-N) expression. (C), AML12 cells were infected with Ad-GFP or Ad-ERRα for 24 hr. Western blot analyses shows CREBH-N expression. (D,E), Ad-GFP or Ad-ERRγ were injected via tail vein into male C57BL/6J mice (n = 5 per group). Following completion of the experiments, mice were sacrificed, and liver tissues were obtained for (D), qRT-PCR analyses to quantify CREBH mRNA levels, and (E), western blot analyses of CREBH-N expression. (F,G), HepG2 cells were infected with Ad-GFP or Ad-ERRγ for 24 hr. (F),Total RNA was isolated for qRT-PCR analyses to quantify CREBH mRNA levels using CREBH primers. (G), Western blot analyses shows CREBH-N expression. Data are representative of three independently performed experiments and shown as mean±SD; *, P<0.05 using Student’s t-test.
Figure 2.
ER stress induces CREBH gene expression via ERRγ.
(A), Activation of the mouse CREBH promoter by ERRγ. Transient transfection was performed in 293T cells with the indicated plasmid DNAs. (B), Activation of the human CREBH promoter by ERRγ. Transient transfection was performed in 293T cells with the indicated plasmid DNAs. (C), Effect of ERRγ knock down on the mouse CREBH promoter activation by ER stress. Transient transfection was performed in 293T cells with the indicated plasmid DNAs. (D, E), AML12 cells were treated with DMSO or Tm (5µg/mL) alone for 12 hr. or first infected with Ad-shERRγ, and at 48 hr. after infection treated with Tm (5µg/mL) for 12 hr. (D), Total RNA was isolated for qRT-PCR analyses to quantify CREBH mRNA levels using CREBH primers, and (E), Western blot analyses shows ERRγ and CREBH-N expression. Data are representative of three independently performed experiments and shown as mean±SD; *, P<0.05, and **, P<0.005 using Student’s t-test.
Figure 3.
ERRγ regulates activation of CREBH gene promoter.
(A), PGC1α-dependent activation of the mouse CREBH promoter by ERRγ. Transient transfection was performed in 293T cells with the indicated plasmid DNAs. (B), Deletion constructs of the CREBH promoter demonstrate the ERRγ binding site in 293T cells. Transient transfection was performed in 293T cells with the indicated plasmid DNAs. (C), ERRE-dependent activation of the CREBH promoter in 293T cells. 293T cells were transfected with the wild-type or ERRE-mutant CREBH promoter along with ERRγ plasmid DNAs. (D), ChIP assay shows the binding of ERRγ and PGC1α to the endogenous CREBH promoter by semiquantitative PCR. AML12 cells were treated with DMSO or Tm (5µg/mL) for 12 hr. After completion of the treatment, chromatin fragments were prepared and immunoprecipitated with ERRγ, PGC1α, or IgG control antibodies. DNA fragments covering –766 to –642 and –195 to –87 elements on the CREBH promoter were PCR amplified. 10% of the soluble chromatin was used as input. (E), ChIP assay for detection of histone acetylation at the ERRγ/PGC1α binding site under the indicated conditions in AML12 cells. Chromatin fragments were prepared and immunoprecipitated with Acetyl-Histone 3 and Acetyl-Histone 4 antibodies. DNA fragments covering –195 to –87 element on the CREBH promoter were qPCR-amplified as described in the ‘Materials and Methods’ section. Data are representative of three independently performed experiments and shown as mean±SD; *P<0.05, **P<0.005, and ***, p<0.0005 using Student’s t-test.
Figure 4.
ERRγ regulates CREBH target gene.
(A), ERRγ dependent activation of the CRP promoter by ER stress. Transient transfection was performed in 293T cells with the indicated plasmid DNAs. (B), AML12 cells were infected with Ad-GFP or Ad-ERRγ for 24 hr. or first infected with Ad-CREBHi, and at 48 hr. after, infected with Ad-ERRγ for 24 hr. (Left panel), total RNA was isolated for qRT-PCR analyses to quantify CRP mRNA levels using CRP primers, and (right panel), western blot analyses shows CRP expression. (C), AML12 cells were treated with DMSO or Tm (5µg/mL) alone for 12 hr. or first infected with Ad-shERRγ, and at 48 hr. after infection, treated with Tm (5µg/mL) for 12 hr. (Left panel), Total RNA was isolated for qRT-PCR analyses to quantify CRP mRNA levels using CRP primers, and (right panel), Western blot analyses shows CRP expression. Data are representative of three independently performed experiments and shown as mean±SD; *, p<0.05, and **, P<0.005 using Student’s t-test.
Figure 5.
ERRγ inverse agonist inhibits CREBH and CRP in vivo.
(A), GSK5182 was administered prior to (40 mg/kg, p.o. in 30% PEG400/DW) and at 30 minutes after Tm (1 mg/kg, i.p. in 1% DMSO/DW) was administered into C57BL/6J mice (n = 5 per group). Following completion of the experiments, mice were sacrificed, and liver tissues were obtained for western blot analyses of CRP, CREBH-N, and ERRγ. (B), GSK5182 was administered (40 mg/kg, p.o. in 30% PEG400/DW) into C57BL/6J mice (n = 5 per group) or EtOH was administered into C57BL/6J mice (n = 5 per group) or first GSK5182 was administered and then EtOH was administered (n = 5 per group). After completion of the treatment, mice were sacrificed. Western blot analyses shows CRP, CREBH-N and ERRγ expression in liver tissue. (C), GSK5182 was administered (40 mg/kg, p.o. in 30% PEG400/DW) into db/db mice (n = 5 per group) for 30 days. After completion of the treatment, mice were sacrificed. Western blot analyses shows CRP, CREBH-N and ERRγ expression in liver tissue. (D), Schematic representation of the proposed model in which ER stress mediated induction of CREBH is mediated through ERRγ.