Serum cholesterol selectively regulates glucocorticoid sensitivity through activation of JNK

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   Figure 1

   Serum factors induce Gc resistance. HeLa cells were transiently transfected with MMTV-Luc, Renilla-Luc and GR (A and B) together with pcDNA5 or SRC1 expression vectors (C and D). Following overnight culture in either 10 or 50% serum, cells were treated with hydrocortisone (HC) (A and C) or dexamethasone (Dex, 0–100 nM) (B and D) for 16 h before luciferase assay. Cells were cultured in either 10 or 50% serum, treated with 100 nM Dex for up to 24 h before immunoblot for GR and ^PS211-GR (E). Tubulin was used as a loading control. Cells were transfected with GR–GFP and then cultured in either 10 or 50% serum overnight. Cells were treated with 100 nM Dex and imaged in real time. Images were acquired every 5 min. Average time for GR nuclear translocation in either 10 or 50% serum was calculated and presented in (F). Representative images are shown (G). Scale bar, 50 μM. Experiments were performed in triplicate and repeated three times. Graphs show mean±s.e.m. *P<0.05.
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   Figure 2

   Serum impairs GR transactivation in a reversible manner. HeLa cells were cultured in either 10 or 50% serum overnight, then treated with 100 nM Dex for 4 h before RNA purification. Transcripts of four well-characterised GR transactivated endogenous genes FK506 binding protein 5 (FKBP5), metallothionein 1X (MT1X), glucocorticoid-induced leucine zipper (GILZ), and period circadian protein homolog 1 (PER1) were measured by qRT-PCR (all normalised to GAPDH) (A, B, C and D). Cells were either continually cultured in either 10 or 50% serum, or cultured in 50% serum then returned to 10% serum as shown in (E) before treatment with 100 nM Dex for 4 h and transcript analysis for MT1X (F), FKBP5 (G), GILZ (H), and PER1 (I). Experiments were performed in triplicate and repeated three times. Graphs show mean±s.e.m. *P<0.01 and **P<0.001. NS, not significant.
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   Figure 3

   Serum increases GR transrepression of IL6 through AP1 elements. HeLa cells were transiently transfected with TAT3-Luc (A) or pHH-Luc (B), together with Renilla-Luc and GR. 24 h later, cells were transferred to culture in either 10 or 50% serum then treated with Dex for 16 h before luciferase assay. HeLa cells were cultured in either 10 or 50% serum overnight, and then treated with either 100 nM Dex (C) or 100 μM ZnSO₄ (D) for 4 h before RNA purification and MT1X transcript measured by qRT-PCR (normalised to GAPDH). Cells were cultured in 10 or 50% serum overnight then treated with 5 ng/ml TNF for 1 h and Dex for a further 4 h. RNA was extracted and IL6 transcript measured by qRT-PCR (normalised to GAPDH) (E). HeLa cells were transiently transfected with WT or AP1 deletant IL6-Luc plasmids then cultured in 10 or 50% serum. Cells were treated with 100 nM PMA and 100 nM Dex for 16 h and then lysed for luciferase assay (F). Graphs show mean±s.e.m. Experiments were carried out in triplicate and repeated three times. *P<0.05, **P<0.01, and ***P<0.001. NS, not significant.
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   Figure 4

   Serum impairs Gc action through activation of JNK. HeLa cells were transiently transfected with MMTV-Luc and GR overnight and then cultured in 10 or 50% serum. The cells were treated with Latrunculin B (A) or MAP kinase inhibitors (B) (ERK PD98059, p38 SB203580, and JNK SP600125) together with 100 nM Dex for 6 h before luciferase assay. The cells were transiently transfected with MMTV-Luc and GR, together with JNK (C) or ERK (D) expression vectors (or pcDNA5 as a control) overnight. The cells were transferred to 10 or 50% serum, treated with 100 nM Dex and 10 μM JNK inhibitor (SP600125) or ERK inhibitor (PD98059) for 6 h and then assayed for luciferase activity. HeLa cells were transfected with JNK or ERK expression vectors (or pcDNA5 as a control), cultured in 10 or 50% serum, and then treated with 100 nM Dex for 4 h before RNA extraction. Expression of MT1X (E), FKBP5 (F), GILZ (G), and PER1 (H) transcripts were measured by qRT-PCR (all normalised to GAPDH). The experiments were carried out in triplicate and repeated three times. Graphs show mean±s.e.m. *P<0.05. NS, not significant.
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   Figure 5

   Modulation of cellular cholesterol regulates Gc effects. HeLa cells were transiently transfected with MMTV-Luc reporter gene and GR then transferred to 10 or 50% serum overnight. Cells were treated with Genistein (as indicated) for 4 h, and then treated with 100 nM Dex for a further 4 h before lysis and luciferase assay (A). Cells were also cultured in either normal serum, charcoal stripped serum or lipoprotein-deficient serum (B and C), and then treated with 100 nM Dex for 16 h before luciferase assay. Cells were cultured in 10% serum with 10 μg/ml cholesterol (CHOL) complexed with 0.5% methyl-β-cyclodextrin (MCD) or with 0.5% MCD alone (D). Cells transfected with MMTV-Luc and GR, cultured in 10 or 50% serum were treated with Simvastatin (as indicated) together with 100 nM Dex for 16 h before luciferase assay (E). Cells were cultured in 50% serum, together with cholesterol complexed with 0.5% MCD, 0.5%, or 5% MCD alone, then treated with 100 nM Dex for 16 h before luciferase assay (F). Experiments were carried out in triplicate and repeated three times. Graphs show mean±s.e.m. *P<0.05 and **P<0.01.
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   Figure 6

   Cholesterol regulates GR transactivation via activation of JNK. HeLa cells were cultured in 10% serum or 10% serum supplemented with 10 μg/ml cholesterol overnight (A and B), or with 50% serum or 50% serum supplemented with 5% MCD (C and D). Cells were harvested after 4 h treatment with either 100 nM Dex or DMSO and analysed by qRT-PCR for expression of MT1X and FKBP5 (normalised to GAPDH). HeLa cells cultured in 10% serum supplemented with 10 μg/ml cholesterol (CHOL) or 50% serum supplemented with 5% MCD overnight, and then protein lysates analysed by immunoblot for JNK and phosphorylated JNK (E). Tubulin and caveolin expression was used as loading controls. Cells were cultured in 10% serum supplemented with 10 μg/ml cholesterol, together with 10 μM JNK inhibitor (SP600125) for 6 h. Following treatment with 100 nM Dex for an additional 4 h, expression of GR transactivation of either MT1X (F) or FKBP5 (G) was measured by qRT-PCR (normalised to GAPDH). Graphs show average±s.e.m. Experiments were carried out in triplicate and repeated three times. *P<0.05 and **P<0.001.
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   Figure 7

   Cholesterol selectively regulates GR transactivation in vivo. Serum samples from WT and ApoE^−/− mice (eight animals per group) were analysed for levels of cholesterol, HDL and triglycerides (A). Morning (0700 h) and evening (1900 h) serum samples from ApoE^−/− mice (16 animals per group) were assayed for corticosterone (B). WT and ApoE^−/− mice were subjected to a dexamethasone (Dex) suppression test. Mice (eight animals per group) were given i.p. injection of Dex (1 mg/kg) and then serum collected 4 h later (1900 h). Serum samples were assayed for corticosterone (C). WT and ApoE^−/− mice (five animals per group) were given i.p. injection of Dex (1 mg/kg) and then livers collected 4 h later. Livers were analysed for GR expression by immunoblot (D). Blots show samples from three different animals. Livers were also analysed for induction of GR target genes MT1, PER1, and FKBP5 (E), DUSP1, NFKBIA, and GILZ (F) by qRT-PCR (all normalised to GAPDH). Graphs show mean±s.e.m. *P<0.05 and **P<0.01. NS, not significant.

• © 2014 The authors