Hepatic FOXO1 Target Genes Are Co-regulated by Thyroid Hormone via RICTOR Protein Deacetylation and MTORC2-AKT Protein Inhibition - PubMed (original) (raw)
Hepatic FOXO1 Target Genes Are Co-regulated by Thyroid Hormone via RICTOR Protein Deacetylation and MTORC2-AKT Protein Inhibition
Brijesh K Singh et al. J Biol Chem. 2016.
Abstract
MTORC2-AKT is a key regulator of carbohydrate metabolism and insulin signaling due to its effects on FOXO1 phosphorylation. Interestingly, both FOXO1 and thyroid hormone (TH) have similar effects on carbohydrate and energy metabolism as well as overlapping transcriptional regulation of many target genes. Currently, little is known about the regulation of MTORC2-AKT or FOXO1 by TH. Accordingly, we performed hepatic transcriptome profiling in mice after FOXO1 knockdown in the absence or presence of TH, and we compared these results with hepatic FOXO1 and THRB1 (TRβ1) ChIP-Seq data. We identified a subset of TH-stimulated FOXO1 target genes that required co-regulation by FOXO1 and TH. TH activation of FOXO1 was directly linked to an increase in SIRT1-MTORC2 interaction and RICTOR deacetylation. This, in turn, led to decreased AKT and FOXO1 phosphorylation. Moreover, TH increased FOXO1 nuclear localization, DNA binding, and target gene transcription by reducing AKT-dependent FOXO1 phosphorylation in a THRB1-dependent manner. These events were associated with TH-mediated oxidative phosphorylation and NAD(+) production and suggested that downstream metabolic effects by TH can post-translationally activate other transcription factors. Our results showed that RICTOR/MTORC2-AKT can integrate convergent hormonal and metabolic signals to provide coordinated and sensitive regulation of hepatic FOXO1-target gene expression.
Keywords: AKT; MTORC2; RICTOR; carbohydrate metabolism; gene regulation; gluconeogenesis; insulin signaling; thyroid hormone; transcription coregulator.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Figures
FIGURE 1.
Thyroid hormone (T3)-regulated gene expression of a subset of Foxo1 target genes. A, Western blot analysis to confirm Foxo1 knockdown in mouse liver tissue. Lane I, control siRNA injected euthyroid (EuTH) mice; lane II, control siRNA injected hyperthyroid (HyperTH) mice; lane III, Foxo1 siRNA injected HyperTH mice. B, gene ontology-KEGG Pathway analysis of the genes obtained by custom Foxo1-ChIP-Seq genes (as described under “Experimental Procedures”) indicating nominal significance and false discovery rate. C, Venn diagram for overlap analysis of core-enriched significantly differentially expressed genes under custom Foxo1-ChIP-Seq gene pathway data set in HyperTH versus EuTH (TH); HyperTH versus Foxo1 knockdown + HyperTH (Foxo1). Venn diagram was formed as described under “Experimental Procedures” by integrating the data from core-enriched genes from gene expression microarray data. Green circle shows Foxo1 up-regulated genes (75 genes). Red circle shows TH up-regulated genes (37). Brown circle shows TH-Foxo1 co-regulated genes (27). D, overlap analysis of TH-only regulated (10) and TH-Foxo1 co-regulated genes (27) in the liver tissues from HyperTH versus EuTH (TH) and HyperTH versus Foxo1 knockdown + HyperTH mice (Foxo1); and the genes have THRB1 binding to their locus (Thrb1, black circle). The data were obtained by comparisons from transcriptome analysis, Foxo1-ChiP-Seq, and THRB1-Chip-seq analysis. Venn diagram was formed as described under “Experimental Procedures” by integrating the data from core-enriched genes from gene expression microarray data. Brown circle shows TH-Foxo1 co-regulated genes (16 + 11 = 27). Red circle shows TH regulated genes (8 + 2 = 10). Black circle shows THRB1 binding in the genes (2 + 16 = 18). E, transcript expression validation by RT-qPCR for T3 as well as Foxo1 target genes in the liver tissues of mice as described in A. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.E. F and G, transcript expression validation by RT-qPCR for T3 alone (G) or Foxo1 alone (H) regulated genes in the liver tissues of mice as described in A. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.E. H, transcript expression by RT-qPCR for T3 and Foxo1 target genes in the primary mouse hepatocytes and human hepatic cell line HepG2 expressing THRB1. Statistical significance was calculated as *, p < 0.05, and error bars represent ±S.D. NS, not significant.
FIGURE 2.
Thyroid hormone (T3)-regulated gene expression of PCK1 and IGFBP1 in a FOXO1-dependent manner. A and B, FOXO1 knockdown and overexpression in HepG2 cells expressing control vector containing no receptor (NR-HepG2) or THRB1 (THRB1-HepG2) confirmed by Western blotting. ACTB, β-actin. Overexpression of wild type FOXO1 (FLAG-FOXO1) as well as phosphorylation mutant of FOXO1 (FLAG-_FOXO1_AAA) (T24A/S256A/S319A) in these HepG2 cells was performed. C and E show results for NR-HepG2 cells, ands D and F show results for THRB1-HepG2 cells. C and D show PCK1 and IGFBP1 transcripts expression by RT-qPCR in NR-HepG2 and THRB1-HepG2, respectively, under FOXO1 knocked down conditions. Data were calculated by comparing with siRNA control in panels C and D. T3 was used at 100 nm concentration. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. E and F, FOXO1 target genes PCK1 and IGFBP1 transcripts expression by RT-qPCR during FOXO1 overexpression in NR-HepG2 and THRB1-HepG2, respectively. Data were calculated by comparing with empty vector in panels E and F. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. NS, not significant.
FIGURE 3.
Thyroid hormone (T3) increased FOXO1 nuclear localization and inhibited AKT phosphorylation. A, immunofluorescence confocal microscopy analysis of FOXO1 nuclear localization. FOXO1 was stained with Alexa Fluor® 594 secondary antibody (Molecular Probes, Invitrogen) and nucleus with DAPI and then given pseudo colors (red for nucleus and green for FOXO1) for better overlay visualization in ZEN 2012 SP1 (black edition; Carl Zeiss) software. Images were captured at ×40 magnification. Image shown in inset is enlarged view of indicated parent image. B, quantification of confocal microscopy data for the ratio of nuclear FOXO1 to total FOXO1 in HepG2 cells by computing percent co-localization, with or without T3 (100 n
m
) treatment. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. C, Western blot analysis of cytoplasmic and nuclear fractions to estimate FOXO1 nuclear localization and AKT/FOXO1 phosphorylation after T3 treatment in THRB1-HepG2 cells at given time points. GAPDH was used as loading control for cytosolic fraction and histone H3 for nuclear fraction. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D.
FIGURE 4.
Thyroid hormone (T3) inhibited AKT-dependent phosphorylation and increased FOXO1 target gene expression. A–C, Western blot analysis to analyze phosphorylated Akt and Foxo1 in liver tissues (A) from euthyroid (EuTH) and hyperthyroid (HyperTH) mice, primary mouse hepatocytes (B) and HepG2 cells expressing THRB1 (C). Bar graphs below each Western blot data set represent relative densitometric measurements. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. D and E, Western blot analysis showing dose response (D) and time course (E) for T3 treatment on phosphorylated AKT and FOXO1 in THRB1-HepG2 cells. Line graphs below each Western blot data set represent relative densitometric measurements. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. ACTB, β-actin. F and G, graph showing dose response (F) and time course (G) of FOXO1 target genes PCK1 and IGFBP1 transcript expression by RT-qPCR in THRB1-HepG2, respectively. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D.
FIGURE 5.
Thyroid hormone (T3) decreased AKT/FOXO1 phosphorylation and increased FOXO1 target gene expression in THRB1-dependent manner. A and B, Western blot analysis to confirm Thrb1 knock-out (A) in mouse liver tissues and phosphorylated Akt/Foxo1 (B) in liver tissues from hypothyroid (HypoTH) and hyperthyroid (HyperTH) of Thrb+/+ and _Thrb_−/− mice, respectively. Bar graphs below each Western blot data set represent relative densitometric measurements. Statistical significance was calculated as *, p < 0.05, and error bars represent ± S.E. C and D, Western blot analysis to confirm stable overexpression of THRB1 in HepG2 cells (C) and analysis of phosphorylated AKT.FOXO1 in NR-HepG2 and THRB1-HepG2 cells (D). Bar graph represents relative densitometric measurements of phosphorylated AKT and FOXO1. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. E, transcript expression of FOXO1 target genes (PCK1 and IGFBP1) by RT-qPCR analysis in NR-HepG2 Versus HepG2 cells expressing THRB1. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D.
FIGURE 6.
Thyroid hormone (T3) induced FOXO1 target genes in an AKT-dependent manner. A, Western blot analysis for phosphorylated AKT and FOXO1 in HepG2 cells. LY292004 compound (5 μ
m
) was used to inhibit AKT in HepG2 cells along with or without T3 (100 n
m
). Bar graph represents relative densitometric measurements of phosphorylated AKT and FOXO1. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. B, transcript expression of PCK1 and IGFBP1 in HepG2 cells treated with LY292004 compound and/or T3. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. C, Western blot analysis of myristoylated AKT (myrAKT), a constitutively active form of AKT, overexpression in THRB1-HepG2 cells. Bar graph represents relative densitometric measurements of total as well as phosphorylated AKT. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. D, transcript expression of PCK1 and IGFBP1 in HepG2 cells overexpressing myrAKT and treated with or without T3. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. ACTB, β-actin.
FIGURE 7.
RICTOR was required for thyroid hormone (T3)-induced FOXO1 target genes. A, Western blot analysis for RICTOR knockdown in THRB1-HepG2 cells and phosphorylation of AKT as well as FOXO1. Bar graph represents relative densitometric measurements of phosphorylated AKT and FOXO1. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. B, RT-qPCR analysis was performed to measure PCK1 and IGFBP1 transcript expression in RICTOR knockdown THRB1-HepG2 cells with/without T3 (100 n
m
). Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. C, Western blot analysis for RICTOR overexpression in HepG2 cells and AKT phosphorylation. Bar graph represents relative densitometric measurements of phosphorylated AKT. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. D, PCK1 and IGFBP1 transcripts expression in THRB1-HepG2 cells overexpressing RICTOR and treated with or without T3. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. E–G, immunoprecipitation-Western blot (IP-WB) analysis to observe RICTOR acetylation in NR-HepG2 versus THRB1-HepG2 cells (E), liver tissues (F) from euthyroid (EuTH) and hyperthyroid (HyperTH) mice and from Thrb+/+ and _Thrb_−/− mice (G), respectively. IgG-heavy chain Western blot was used to normalize acetylated lysine pulldown in E–G. Bar graph represents relative densitometric measurements for acetylated RICTOR. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. (E) or mean ± S.E. (F and G). ACTB, β-actin; NS, not significant.
FIGURE 8.
Thyroid hormone (T3)-induced RICTOR deacetylation was SIRT1-dependent. A, IP-WB analysis to observe RICTOR acetylation in SIRT1 knockdown NR-HepG2 versus THRB1-HepG2 cells treated with or without T3. B, IP-WB analysis to observe Rictor acetylation in liver tissues from euthyroid (EuTH), hyperthyroid (HyperTH), and Ex527-treated hyper-TH mice. IgG-heavy chain Western blot was used to normalize acetylated lysine pulldown in A and B. Bar graph represents relative densitometric measurements for acetylated RICTOR. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. (A) or mean ± S.E. (B). C, co-IP-WB of FLAG-tagged SIRT1 to observe SIRT1 interaction with MTORC2 components (MTOR and RICTOR) in THRB1-HepG2 cells treated with or without T3. Bar graph represents relative densitometric measurements for MTOR and RICTOR normalized to total SIRT1 pulled down. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. D, NAD+/NADH measurement in THRB1-HepG2 cells treated with or without T3. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. E, measurement of OCR in THRB1-HepG2 cells treated with or without T3 for mentioned time points using Seahorse extracellular flux analyzer. Oligomycin (Oligo.; 1 μ
m
; F0F1-ATP synthase inhibitor), FCCP (1 μ
m
; mitochondrial uncoupler) and rotenone (R; 1 μ
m
) + antimycin A (A; 1 μ
m
) (complex I and complex III inhibitors in electron transport chain, respectively) was used to analyze various parameters of mitochondrial OCR. F, computational analysis of functional parameters of mitochondrial OCR as under “Experimental Procedures,” and the data were plotted as a bar graph. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.E.
FIGURE 9.
SIRT1 inhibition inversely affects thyroid hormone (T3)-dependent decrease in AKT/FOXO1 phosphorylation and FOXO1 activation. A, Western blot analysis of Akt as well as Foxo1 phosphorylation in liver tissues from euthyroid (EuTH), hyperthyroid (HyperTH), and Ex527-treated HyperTH mouse. Ex527 compound was used to inhibit Sirt1 activity in vivo. Bar graph represents relative densitometric measurements of phosphorylated Akt and Foxo1. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.E. B, Western blot analysis of SIRT1 knockdown in THRB1-HepG2 cells and phosphorylation and AKT and FOXO1 in SIRT1 knockdown cells treated with or without T3 (100 n
m
). Bar graph represents relative densitometric measurements of phosphorylated AKT and FOXO1. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. C, transcripts expression of FOXO1 target genes PCK1 and IGFBP1 using RT-qPCR in SIRT1 knockdown cells treated with or without T3. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D.
FIGURE 10.
AKT activation inhibited SIRT1-mediated thyroid hormone (T3)-induced dephosphorylation of AKT/FOXO1 and FOXO1 activation. A, Western blot analysis of FLAG-tagged SIRT1 overexpression and AKT/FOXO1 phosphorylation in THRB1-HepG2 cells treated with or without T3 (100 n
m
). Bar graph represents relative densitometric measurements of phosphorylated AKT and FOXO1. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D. B, PCK1 and IGFBP1 transcripts expression after co-overexpression of FLAG-SIRT1 and myrAKT in these HepG2 cells with or without T3. Statistical significance was calculated as *, p < 0.05, and error bars represent mean ± S.D.
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