Class 3 phosphoinositide 3-kinase promotes hepatic glucocorticoid receptor stability and transcriptional activity

Abstract

Aim: Lipid kinase class 3 phosphoinositide 3-kinase (PI3K) and nuclear receptor transcription factor glucocorticoid receptor (GR) play essential physiological roles in metabolic adaptation to fasting by activating lysosomal degradation by autophagy and metabolic gene expression, yet their functional interaction is unknown. The requirement of class 3 PI3K for GR function was investigated in liver tissue.

Methods: Inactivation of class 3 PI3K was achieved through deletion of its essential regulatory subunit Vps15, by expressing Cre-recombinase in the livers of Vps15^f/f mice. The response to both 24-h fasting and synthetic GR ligand, dexamethasone (DEX) was evaluated in control and mutant mice. Liver tissue was analysed by immunoblot, RT-qPCR, and LC-MS.

Results: Vps15 mutant mice show decreased transcript levels of GR targets, coupled with lower nuclear levels of total and phosphorylated on Ser211, GR protein. Acute DEX treatment and 24-h fasting both failed to re-activate expression of GR targets in the livers of Vps15 mutant mice to the levels observed in controls. Decreased levels of endogenous GR ligand corticosterone and lower expression of 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), a metabolic enzyme that controls corticosterone availability, were found in the livers of Vps15 mutants. Hepatic Vps15 depletion resulted in the activation of nuclear Akt1 signalling, which was paralleled by increased polyubiquitination of GR.

Conclusion: In the liver, class 3 PI3K is required for corticosterone metabolism and GR transcriptional activity.

Keywords: Akt signalling; class 3 phosphoinositide 3-kinase; corticosterone; glucocorticoid receptor; liver.

FIGURE 1

Repressed transcriptional activity of GR in hepatic mutants of Vps15. A, Venn diagram showing the overlap of the differentially expressed genes in the liver in microarray sets of GR‐LKO (GSE75682) and Vps15‐LKO (MTAB‐7685) mice. The numbers of unique genes modified above 1.2‐fold in each microarray and overlap between two data sets are shown. Functional clustering analysis of significantly enriched biological processes by DAVID‐GO for co‐regulated genes (downregulated (B) or upregulated (C)) in the livers of GR‐LKO and in Vps15‐LKO mice. D, Transcript levels by RT‐qPCR of GR target genes functioning in different processes. The liver tissue of random‐fed, 6‐week‐old Vps15^f/f and AlbCre⁺; Vps15^f/f mice was collected in the middle of the day corresponding to physiological fasting period. Data are means ± SEM (n = 4 for Vps15^f/f, n = 4 for AlbCre⁺; Vps15^f/f, *P < .05, ^† P <.01, ^‡ P <.001: vs Vps15^f/f mice, two‐tailed, unpaired Student's t test)

FIGURE 2

Decreased nuclear expression of GR in the livers of Vps15‐LKO mice. A, Immunoblot analysis of total protein of liver extracts from random‐fed, 6‐week‐old Vps15^f/f and AlbCre⁺; Vps15^f/f mice using indicated antibodies. The liver tissue was collected in the middle of the day corresponding to physiological fasting period. Immunoblot with anti‐GAPDH antibody served as a loading control. Densitometric analyses of protein levels normalized to GAPDH levels presented as folds over Vps15^f/f mice. Data are means ± SEM (n = 4 for Vps15^f/f, n = 4 for AlbCre⁺; Vps15^f/f, *P <.05, ^† P <.01, ^‡ P <.001: vs Vps15^f/f, two‐tailed, unpaired Student's t test). B, Relative transcript levels of GR isoforms, GRα and GRβ in the livers of Vps15^f/f and AlbCre⁺; Vps15^f/f analysed by RT‐qPCR (liver tissue collected as in (A). Data are means ± SEM (n = 8 for Vps15^f/f, n = 9 for AlbCre⁺; Vps15^f/f, *P <.05: vs Vps15^f/f mice, two‐tailed, unpaired Student's t test). C, Immunoblot analysis using indicated antibodies of nuclear fraction of liver extracts from Vps15^f/f and AlbCre⁺; Vps15^f/f mice (liver tissue collected as in (A). Immunoblot with β‐catenin antibody served as a loading control. Densitometric analyses of protein levels normalized to β‐catenin levels presented as folds over Vps15^f/f. Data are means ± SEM (n = 4 for Vps15^f/f, n = 4 for AlbCre⁺; Vps15^f/f, ^‡ P <.001: vs Vps15^f/f, two‐tailed, unpaired Student's t test)

FIGURE 3

Incomplete reactivation of GR in the livers of Vps15‐LKO mice in response to fasting and acute DEX treatment. A, Relative transcript levels of GR target genes in the livers of Vps15^f/f and AlbCre⁺; Vps15^f/f by RT‐qPCR. The 6‐week‐old mice were fasted at the onset of night (active feeding period) and both treatment groups sacrificed 24 h after. Data are means ± SEM (Vps15^f/f (n = 6 for fed and fast group), AlbCre⁺; Vps15^f/f (n = 4 and n = 5 for fed and fast group), *P <.05, ^† P <.01, ^‡ P <.001: two‐tailed, unpaired Student's t test). B, Relative transcript levels of GR target genes in the livers of Vps15^f/f and AlbCre⁺; Vps15^f/f mice by RT‐qPCR. The 6‐week‐old mice were injected IP with DEX (50 mg/kg) at the onset of the day phase and liver tissue collected 5 h later (middle of the day corresponding to physiological fasting period). Data are means ± SEM (Vps15^f/f (n = 4 for placebo and DEX group), AlbCre⁺; Vps15^f/f (n = 5 for placebo and DEX group), *P <.05, ^† P <.01, ^‡ P <.001: two‐tailed, unpaired Student's t test). C, Immunoblot analysis of nuclear extracts of the liver tissue collected under the conditions as in (A) and (B) using indicated antibodies. Densitometric analyses of protein levels normalized to β‐catenin levels presented as folds over Vps15^f/f‐placebo condition. Data are means ± SEM (Vps15^f/f (n = 4 for fed and fast group), AlbCre⁺; Vps15^f/f (n = 4 for fed and fast group), Vps15^f/f (n = 3 and n = 4 for placebo and DEX group), AlbCre⁺; Vps15^f/f (n = 3 and n = 4 for placebo and DEX group), *P <.05, ^† P <.01, two‐tailed, unpaired Student's t test)

FIGURE 4

Decreased corticosterone levels and increased GR polyubiquitination in the livers of Vps15‐LKO mice. A, Immunoblot analysis of total protein extracts of the liver tissue of random‐fed, 6‐week‐old Vps15^f/f and AlbCre⁺; Vps15^f/f using indicated antibodies. The liver tissue was collected in the middle of the day corresponding to the physiological fasting period. Densitometric analyses of protein levels normalized to GAPDH protein levels presented as folds over Vps15^f/f. Data are means ±SEM (n = 4 for Vps15^f/f, n = 4 for AlbCre⁺; Vps15^f/f, ^† P <.01: vs Vps15^f/f, two‐tailed, unpaired Student's t test). B, Immunoblot analysis of nuclear extracts of the liver tissue collected as in (A) using indicated antibodies. Densitometric analyses of protein levels normalized to β‐catenin levels presented as folds over Vps15^f/f. Data are means ± SEM (n = 4 for Vps15^f/f, n = 4 for AlbCre⁺; Vps15^f/f, ^† P <.01: vs Vps15^f/f, two‐tailed, unpaired Student's t test). C, Relative transcript levels of 11βHSD1, C/EBPα, and C/EBPβ in the livers Vps15^f/f and AlbCre⁺; Vps15^f/f mice collected as in (A) analysed by RT‐qPCR. Data are means ± SEM (n = 6 for Vps15^f/f, n = 4 for AlbCre⁺; Vps15^f/f, ^† P <.01, ^‡ P <.001: vs Vps15^f/f, two‐tailed, unpaired Student's t test). D, Corticosterone levels in the liver tissue and in plasma of mice collected as in (A), analysed by LC‐MS/MS and presented as peak area (arbitrary units). Data are means ± SEM (n = 5 for Vps15^f/f, n = 7 for AlbCre⁺; Vps15^f/f, ^† P <.01: vs Vps15^f/f, two‐tailed, unpaired Student's t test). E, Immunoblot analysis of nuclear extracts of livers collected as in (A) using indicated antibodies. Densitometric analyses of protein levels normalized to β‐catenin levels presented as folds over Vps15^f/f. Data are means ± SEM (n = 4 for Vps15^f/f, n = 4 for AlbCre⁺; Vps15^f/f, ^† P <.01: vs Vps15^f/f, two‐tailed, unpaired Student's t test). F, Immunoprecipitation of GR from total protein extracts of livers of mice collected as in (A) followed by immunoblot with anti‐ubiquitin antibodies. Densitometric analyses of ubiquitination levels normalized to GR levels presented as folds over Vps15^f/f. Data are means ± SEM (n = 3 for Vps15^f/f, n = 3 for AlbCre⁺; Vps15^f/f, ^† P <.01: vs Vps15^f/f, two‐tailed, unpaired Student's t test)