Glucocorticoid Receptor Accelerates, but Is Dispensable for, Adipogenesis

Abstract

Dexamethasone (DEX), a synthetic ligand for glucocorticoid receptor (GR), is routinely used to stimulate adipogenesis in culture. GR-depleted preadipocytes show adipogenesis defects 1 week after induction of differentiation. However, it has remained unclear whether GR is required for adipogenesis in vivo By deleting GR in precursors of brown adipocytes, we found unexpectedly that GR is dispensable for brown adipose tissue development in mice. In culture, GR-deficient primary or immortalized white and brown preadipocytes showed severely delayed adipogenesis 1 week after induction of differentiation. However, when differentiation was extended to 3 weeks, GR-deficient preadipocytes showed levels of adipogenesis marker expression and lipid accumulation similar to those of the wild-type cells, indicating that DEX-bound GR accelerates, but is dispensable for, adipogenesis. Consistently, DEX accelerates, but is dispensable for, adipogenesis in culture. We show that DEX-bound GR accelerates adipogenesis by directly promoting the expression of adipogenic transcription factors CCAAT/enhancer-binding protein alpha (C/EBPα), C/EBPβ, C/EBPδ, KLF5, KLF9, and peroxisome proliferator-activated receptor γ (PPARγ) in the early phase of differentiation. Mechanistically, DEX-bound GR recruits histone H3K27 acetyltransferase CBP to promote activation of C/EBPβ-primed enhancers of adipogenic genes. These results clarify the role of GR in adipogenesis in vivo and demonstrate that DEX-mediated activation of GR accelerates, but is dispensable for, adipogenesis.

Keywords: GR; adipogenesis; dexamethasone; glucocorticoid receptor.

FIG 1

GR is largely dispensable for BAT development. GR^f/f mice were crossed with GR^f/+; Myf5-Cre mice to obtain GR^f/f; Myf5-Cre (conditional KO, cKO) and littermate control (GR^f/f, f/f) mice and embryos. (A to E) Characterization of GR^f/f; Myf5-Cre adult mice. (A) Genotyping results. The expected ratios of the four genotypes are 1:1:1:1. (B) Representative pictures of male mice (left panel) and body weight of f/f (n = 9) or cKO (n = 12) mice (right panel). (C) Pictures of isolated adipose tissues. iWAT, interscapular WAT; epi-WAT, epididymal WAT; ing-WAT, inguinal WAT; rWAT, retroperitoneal WAT. (D) Representative pictures of interscapular BAT. (E) Total RNA was extracted from BAT of f/f (n = 9) or cKO (n = 12) mice for qRT-PCR analysis of GR, adipogenesis markers Pparγ, Cebpα, and Fabp4 as well as BAT markers Prdm16 and Ucp1. Quantitative PCR data in all figures are presented as means ± SEM. ***, P < 0.001. n.s., no significance. (F to L) Characterization of GR^f/f; Myf5-Cre E18.5 embryos. (F) Representative pictures of E18.5 embryos. (G) Confirmation of GR deletion in E18.5 BAT by qPCR analysis of genomic DNA. (H) E18.5 embryos were sagittally sectioned along the midline. Sections of the interscapular area were stained with hematoxylin and eosin (H&E). B, BAT; M, muscle. (I) RNA-Seq analysis of BAT collected from two E18.5 GR cKO embryos. Pie chart depicts genes up- or downregulated in cKO samples. The threshold for up- or downregulation is 2-fold. (J) The genome browser view shows the deletion of exon 2 of GR gene in cKO samples. (K) RNA was extracted from E18.5 BAT of f/f (n = 4) or cKO (n = 8) embryos for qRT-PCR analysis. (L) List of the most significantly downregulated mRNAs in E18.5 BAT of GR cKO embryos. Only genes with expression levels with an RPKM of >3.3 in the f/f BATs were included.

FIG 2

Cold tolerance test of GR^f/f; Myf5-Cre mice. (A) Body temperatures of GR^f/f; Myf5-Cre (cKO) and their littermate control (f/f) mice (n = 5 per group) after acute exposure to 4°C for 5 h. Body temperatures were measured every hour. (B) qRT-PCR analysis of thermogenic genes in BAT. RT, room temperature. Data are presented as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n.s., no significance.

FIG 3

GR accelerates, but is largely dispensable for, adipogenesis and induction of master adipogenic TFs. (A to C) GR^f/f immortalized brown preadipocytes were infected with retroviral vector MSCVpuro expressing Cre, followed by brown adipogenesis assays. (A) Whole-cell lysates were subjected to Western blot analysis of GR in preadipocytes. RbBP5 was used as a loading control. Vec, vector. (B) Oil Red O staining of differentiated cells at day 7 (D7) and day 21 (D21). Upper panels, stained dishes; lower panels, representative fields under the microscope. (C) qRT-PCR analysis of gene expression at indicated time points of brown adipogenesis. (D to F) 3T3-L1 white preadipocytes were infected with lentiviral vector expressing control (shControl) or GR knockdown shRNA (shGR), followed by adipogenesis assays. (D) Western blot analysis of GR in preadipocytes. (E) Oil Red O staining of differentiated cells at D7 and D21. (F) qRT-PCR analysis of gene expression.

FIG 4

GR accelerates adipocyte gene induction during 7-day adipogenesis in culture. GR^f/f immortalized brown preadipocytes were infected with MSCVpuro expressing Cre. Cells were collected at the indicated time points during brown adipogenesis for analyses of gene expression using qRT-PCR (A) and RNA-Seq (B to D). (A) Expression of adipogenesis markers at indicated time points. (B and C) Upper panels, schematic pie charts depict GR-dependent and -independent upregulated genes at day 2 (D2) and D7. The threshold for determining up- or downregulation is 2.5-fold. Lower panels, gene ontology (GO) analysis of gene groups defined in upper panels. GO terms with P values of ≤1E−4 for each group of genes are listed. ncRNA, noncoding RNA; acetyl-CoA, acetyl coenzyme A. (D and E) The mRNA levels of GR and Mineralocorticoid receptor (MR) during brown adipogenesis were determined by RNA-Seq. (D) RNA-Seq profiles of GR and MR. (E) RPKM values of GR and MR in GR KO cells. RPKM values indicate gene expression levels.

FIG 5

GR is dispensable for PPARγ- or C/EBPβ-stimulated adipogenesis. GR^f/f immortalized brown preadipocytes were infected with retroviral vector MSCVhygro expressing PPARγ or WZLhygro expressing C/EBPβ. After hygromycin selection, cells were infected with adenoviruses expressing green fluorescent protein (GFP) (Ad-GFP) or Cre (Ad-Cre) for PPARγ-expressing cells or with MSCVpuro expressing vector (Vec) or Cre for C/EBPβ-expressing cells. Adipogenesis were induced for 7 days. (A and D) Western blot analysis for confirming deletion of GR and ectopic expression of PPARγ (A) or C/EBPβ (D) in preadipocytes, respectively. RbBP5 was used as a loading control. (B and E) Oil Red O staining of differentiated cells at D7. (C and F) qRT-PCR analysis of gene expression.

FIG 6

GR accelerates, but is dispensable for, adipogenesis in primary preadipocytes. (A, B) GR^f/f primary white preadipocytes were infected with Ad-GFP or Ad-Cre, followed by white adipogenesis assays until D7 or D21. (A) Oil Red O staining of differentiated cells. (B) qRT-PCR analysis of adipogenesis markers Pparγ, Cebpα, and Fabp4 as well as lipogenic genes Scd1, Srebp1c, Fasn, and Lxr. (C to E) GR^f/f primary brown preadipocytes were infected with Ad-GFP or Ad-Cre, followed by adipogenesis until D7 or D21. (C) Oil Red O staining of differentiated cells. (D) qRT-PCR analysis of GR, adipogenesis markers, BAT markers, and lipogenic genes. (E) D21 mature brown adipocytes were treated with 100 nM CL-316,243 for 4 h. The induction of Ucp1 expression was analyzed by qRT-PCR.

FIG 7

DEX accelerates, but is dispensable for, adipogenesis in culture. Adipogenesis was induced by treating the immortalized wild-type brown preadipocyte cell line WT-1 with the adipogenic cocktail with DEX (MDI) or without DEX (MI). (A) Oil Red O staining of differentiated adipocytes at D21. (B) qRT-PCR analysis of adipogenesis markers as well as lipogenic genes.

FIG 8

GR directly activates expression of multiple adipogenic TFs. GR^f/f immortalized brown preadipocytes were infected with MSCVpuro expressing Cre. Cells were collected at 0 h and 4 h after induction of adipogenesis for RNA-Seq. (A) Schematic of identification of GR-dependent and -independent upregulated genes at 4 h. The threshold for up- or downregulation is 2.5-fold. (B) Heat map showing expression changes of the 247 GR-dependent upregulated genes from 0 h to 4 h. (C) qRT-PCR confirmation of GR-dependent upregulation of C/EBPβ, C/EBPδ, Klf5, Klf9, Cebpα, and Pparγ genes in the early phase of adipogenesis. (D) ChIP-Seq profiles of GR binding on gene loci encoding adipogenic TFs at 4 h.

FIG 9

GR recruits H3K27 acetyltransferase CBP to promote activation of C/EBPβ-primed enhancers. (A to E) ChIP-seq analysis of GR, C/EBPβ, phospho-CREB (p-CREB), H3K4me1, and H3K27ac at 4 h after induction of adipogenesis in GR^f/f immortalized brown preadipocytes. (A) Pie chart depicting the genomic distribution of GR binding regions. Of 2,231 GR biding regions, 1,995 (89.4%) are located on active enhancers. (B) Motif analysis of GR binding regions. (C) Venn diagram showing genomic colocalization of GR with C/EBPβ and p-CREB. (D) Heat maps showing genomic colocalization of C/EBPβ, p-CREB, H3K4me1, and H3K27ac with GR on 1,995 GR⁺ active enhancers as defined for panel A. (E) Average profiles of C/EBPβ and p-CREB binding around the center of GR binding active enhancers at 4 h. (F and G) ChIP-seq analyses of H3K27ac and CBP at 4 h after induction of adipogenesis in GR^f/f preadipocytes infected with retroviral Vec or Cre. Average binding profiles of H3K27ac (F) and CBP (G) around the center of GR binding active enhancers are shown. (H) Genome browser view of GR, C/EBPβ, p-CREB, and CBP binding as well as H3K27ac on Cebpδ locus in Vec and Cre cells.