Fig 1.
Celastrol inhibits C/EBPβ activity.
A. QT6 fibroblasts were co-transfected with the luciferase reporter gene p-240luc containing the C/EBP-inducible promoter of the chicken mim-1 gene, the β-galactosidase expression vector pCMVβ and an expression vector for C/EBPβ transfection, the cells were treated with Celastrol as indicated and harvested after 16 hours. The columns show the average luciferase activity normalized to the β-galactosidase activity. Thin lines show standard deviations. Asterisks indicate statistical significance (** p < 0.01, *** p < 0.001, Student’s t-test). C/EBPβ expression is shown at the bottom. B-D. QT6 cells were transfected with expression vectors for chicken or mouse full-length C/EBPβ or chicken C/EBPα and treated with Celastrol as indicated. Cells were harvested after 16 hours and analyzed by northern blotting for expression of MRP126 and S17 mRNAs (top and middle panel) and by western blotting for the expression of C/EBPβ or C/EBPα (bottom panels). E. 3T3-L1 cells were induced to undergo adipocyte differentiation for 7 days in the absence or presence of the indicated concentrations of Celastrol. The top and middle panels show microscopic pictures of the cells at low magnification and after staining with the fluorescent lipid dye Nile Red. The bottom panels show staining of the cells with Oil-Red O. The top and middle panels at the right show microscopic pictures of differentiated cells at higher magnification. The bottom panels on the right side show western blots of C/EBPβ and β-actin expression in undifferentiated (lane 1) and differentiated (lanes 2–4) cells treated without Celastrol (lane 2) or with 0.5 μM (lane 3) and 1 μM (lane 4) Celastrol.
Fig 2.
Celastrol does not affect DNA-binding of C/EBPβ but disrupts its cooperation with p300.
A. A radiolabeled double-stranded oligonucleotide with a consensus C/EBP binding site was incubated without nuclear extract, with nuclear extract from cells transfected with C/EBPβ expression vector and treated with Celastrol as indicated, or with nuclear extract from untransfected cells. Protein-DNA complexes were analyzed by native polyacrylamide gel electrophoresis. The top panel shows a western blot analysis of aliquots of the nuclear extracts stained with antibodies against C/EBPβ. B. QT6 cells transfected with expression vectors for C/EBPβ or a Jun-C/EBPβ hybrid protein and incubated with Celastrol were analyzed by northern blotting for expression of MRP126 and S17 mRNAs. C. QT6 cells transfected with expression vectors for C/EBPβ and p300 were treated with the indicated concentrations of Celastrol, harvested after 16 hours and analyzed by northern blotting for expression of MRP126 and S17 mRNAs. Numbers below the lanes indicate the amounts of MRP126 mRNA relative to the S17 mRNA as control determined by quantification with a phosphor image analyzer. The signals for MRP126 and S17 mRNAs were obtained by sequential hybridization of the same blot with specific radiolabeled probes. D. QT6 cells transfected with expression vectors for C/EBPβ and p300/1751-2379 and cultivated for 24 hours with or without Celastrol. Aliquots of total cell extracts were then analyzed with antibodies against p300, pp300(Ser2280) and C/EBPβ. Black and white arrows mark the un-phosphorylated and highly phosphorylated p300.
Fig 3.
Celastrol disrupts the C/EBPβ-p300 interaction.
A,B. QT6 fibroblasts transfected with the indicated expression vectors were subjected to GFP-trap experiments. Total cell extracts (TCE) and GFP-trap samples (trap) were analyzed by western blotting with antibodies against GFP and p300. In panel B, the cells were also treated with Celastrol. C. QT6 fibroblasts were transfected with the Gal4-dependent reporter gene pG5E4-38luc, the β-galactosidase plasmid pCMVβ and expression vectors for Gal4-C/EBPβ and p300-VP19, as indicated. The transfected cells were incubated for 12 hours with or without Celastrol followed by analysis of luciferase activities. The luciferase activity was first normalized to the β-galactosidase activity. The normalized luciferase activity of the Gal4-C/EBPβ plus p300-VP16 transfected cells or the Gal4-VP16 transfected cells in the absence of Celastrol was then set to 100%. Asterisks indicate statistical significance (*** p < 0.001, Student’s t-test).
Fig 4.
Inhibition of a cysteine-free mutant of C/EBPβ by Celastrol.
A. QT6 fibroblasts were transfected with the indicated expression vectors and treated with Celastrol and β-mercaptoethanol. Cell extracts were then subjected to GFP-trap and analyzed as in Fig 3. B. QT6 cells were transfected with expression vectors for wild-type or cysteine-free C/EBPβ and treated with or without Celastrol. Cells were analyzed by northern blotting for expression of MRP126 and S17 mRNAs as in Fig 1 and Fig 2.
Fig 5.
Disruption of the C/EBPβ-p300 interaction is dependent on cysteine residues in the Taz2 domain of p300.
A. Sequence comparison of the p300 Taz2 domain from human, mouse and chicken. Cysteine and histidine residues involved in coordination of zinc ions and Cys-1789 and Cys-1790 are highlighted. B-D. QT6 fibroblasts transfected with the indicated expression vectors were subjected to GFP-trap experiments and analyzed as in Fig 3. E. Amino acid sequences of different C/EBP family members implicated in Taz2-binding. Box A and Box B refer to α-helical regions assumed to directly interact with the Taz2 domain. F. Partial view of the Taz2 domain and its interaction with Box A and Box B sequences of C/EBPε. The peptide backbones of the Taz2 domain and of CEBPε are shown in brown and turquois, respectively. The position of Cys-1790 is shown. The figure was created from PDB entry 3t92.
Fig 6.
Model of the inhibition of a transcriptional Myb-C/EBPβ-p300 module by Celastrol.