REV-ERBα and GR compete with each other for binding to HSP90, resulting in the destabilization of GR protein. (A) Western blot of whole-cell extracts from NIH3T3 cells transfected with increasing amounts of expression vector for Rev-erbα with indicated antibodies. The lower panel shows the quantification of GR signal (mean±s.d., n=3). **P<0.01 (t-test). (B) Western blot (WB) of HSP90 immunoprecipitates (IP: HSP90) and whole-cell extracts (Input) from NIH3T3 cells transfected with the expression vector for Rev-erbα. The lower panel shows the quantification of the GR and REV-ERBα signal in HSP90 immunoprecipitates normalized to that of HSP90 (mean±s.d., n=3). *P<0.05 (one-way ANOVA with Bonferroni post-test). (C) Western blot of HSP90 immunoprecipitates (IP: HSP90) and whole-cell extracts (Input) from liver of wild-type (WT) and Rev-erbα^−/− mice (Rev−/−). The lower panel shows the quantification of the GR and REV-ERBα signal in HSP90 immunoprecipitates normalized to that of HSP90 (mean±s.d., n=3). **P<0.01, ***P<0.001 (t-test). (D) Western blot of whole-cell extracts from NIH3T3 cells transfected with expression vector for Rev-erbα and treated with cycloheximide (CHX; 100 μg/ml). The middle panel shows the quantification of GR signal. GR half-life with CHX alone is 10 h (R²=0.84, n=4) and with CHX and Rev-erbα it is 5.2 h (R²=0.98, n=4). The lower panel shows the quantification of HSP90 signal. HSP90 half-life with CHX alone is 8.7 h (R²=0.87, n=4) and with CHX and Rev-erbα it is 5.6 h (R²=0.99, n=4). Results are mean±s.d. The P-values given on the figure were calculated with a two-way ANOVA test.

Cellular distribution of REV-ERBα at different times of day. Immunohistochemistry in the liver of wild-type and Rev-Erbα^−/− mice at ZT8 and ZT20. Staining with antibodies recognizing HSP90 (a marker for cytoplasm) and REV-ERBα are in green and red, respectively. Nuclei are stained with DAPI (blue). The pink color indicates nuclear REV-ERBα and the yellow color cytoplasmic REV-ERBα. Note the reversed cellular distribution of REV-ERBα compared to GR (Fig. 4) in wild type. Scale bar: 50 µm.

The change in cellular distribution of GR that is dependent on the time of day is abolished in Rev-erbα^−/− mice. Immunohistochemistry in the liver of wild-type and Rev-Erbα^−/− mice at ZT8 and ZT20. Staining with antibodies recognizing HSP90 (marker for cytoplasm) and GR are in green and red, respectively. Nuclei are stained with DAPI (blue). The pink color indicates nuclear GR and the yellow color cytoplasmic GR. Scale bar: 50 µm.

REV-ERBα and GR reciprocally influence their nuclear localization. (A) Western blot of the cytoplasm and nuclear fraction of the liver of wild-type (WT) and Rev-Erbα^−/− (Rev^−/−) mice at ZT8 and ZT20. The lower panel shows the quantification of signals (mean±s.d., n=4). *P<0.05, ***P<0.001 (one-way ANOVA with Bonferroni post-test). (B) The levels of corticosterone in the plasma of wild-type (black bars) and Rev-Erbα^−/− (white bars) mice revealed by ELISA (n=6, mean±s.d.).

Diurnal interaction of GR and REV-ERBα with HSP90 in liver and sites of interaction on HSP90. (A) Western blot of HSP90 immunoprecipitates (IP: HSP90) (right panel) and whole-cell extracts (Input) from liver cells of wild-type mice. Livers were collected at ZT0, 4, 8, 12, 16 and 20. (B) Pulldown assays with an N-terminal (N, blue), middle (M, red) and C-terminal (C, green) HSP90 fragment fused to GST that had been expressed in bacteria by IPTG induction and purified with gluthatione sepharose resin. The complex-resin–GST proteins were coimmunoprecipitated with either LBD-GR or REV-ERBα, both fused to a His tag, that had been also expressed in bacteria, purified using nickel resin and eluted using imidazole. The pulldown was detected by western blotting. Anti-GST antibodies reveal a 55-kDa GST-N protein, a 70-kDa GST-M protein and a 40-kDa GST-C protein. An anti-His tag antibody reveals a 25-kDa GR-LBD and 75-kDa REV-ERBα protein in the GST-N, GST-M and GST-C fractions, respectively.

REV-ERBα and GR affect target genes of each other in primary cultured hepatocytes. Western blots of primary cultured hepatocytes of wild-type (WT) and Rev-Erbα^−/− (Rev^−/−) mice. Cells were incubated with dexamethasone (1 µM) for 24 h, and culture medium was changed for one with or without 100 mM ethanol. (A) IκBα (IκB) and p-IκBα (p-IκB) specific antibodies reveal an increase in expression of IκBα in Rev-Erbα^−/− cells and an increase of p-IκBα in wild type after ethanol treatment (mean±s.d., n=3). *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA with Bonferroni post-test). (B) GR- and NF-κB-specific antibodies reveal increased expression of GR in Rev-Erbα^−/− hepatocytes, increased nuclear NF-κB signal in wild type cells and a decreased signal of NF-κB in Rev-Erbα^−/− cells after ethanol treatment (mean±s.d., n=3). *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA with Bonferroni post-test). (C) ELISA of TNF in primary cultured hepatocytes from wild-type and Rev-Erbα^−/− mice. Cells were treated with GR or IκBα shRNA lentiviral particles. Scrambled shRNA lentiviral particles (sc) were used as a negative control. At 24 h after transduction, dexamethasone (1 µM) was added and cells were incubated for 24 h, and then the culture medium was changed for one with or without 100 mM ethanol (mean±s.d., n=3). *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA with Bonferroni post-test). (D) Western blot of primary cultured hepatocytes of wild-type mice. Cells were treated with GR shRNA lentiviral particles. Scrambled shRNA lentiviral particles were used as a negative control. The right panel shows the quantification of signals (mean±s.d., n=3). *P<0.05, **P<0.01 (t-test).