DNA binding-dependent glucocorticoid receptor activity promotes adipogenesis via Krüppel-like factor 15 gene expression - PubMed (original) (raw)
DNA binding-dependent glucocorticoid receptor activity promotes adipogenesis via Krüppel-like factor 15 gene expression
Maki Asada et al. Lab Invest. 2011 Feb.
Abstract
Glucocorticoids, such as dexamethasone, have been used as in vitro inducers of adipogenesis. However, the roles of the glucocorticoid receptor (GR) in adipogenesis have not been well characterized yet. Here, we show that inhibition of GR activity using the GR antagonist RU486 prevents human mesenchymal stem cell and mouse embryonic fibroblast (MEF) differentiation into adipocytes. Moreover, in MEFs isolated from GR knockout (GR(null)) and GR(dim) mice deficient in GR DNA-binding activity, adipogenesis was blocked. We identified glucocorticoid response element sites in the first intron of KLF15 by bioinformatical promoter analysis and confirmed their functional relevance by demonstrating GR interaction by chromatin immunoprecipitation. Moreover, transfection of MEFs with siRNA for KLF15 significantly attenuated the expressions of adipogenic-marker genes and the lipid accumulation. Our results provide a new mechanism for understanding glucocorticoids-dependent adipogenesis and that GR promotes adipogenesis via KLF15 gene expression as a transcriptional direct target.
Figures
Figure 1. Effect of GR Antagonist RU486 on Adipogenesis in hMSCs and MEFs
hMSCs were differentiated into adipocytes with AIM medium (containing 500 µM IBMX, 1 µM Dex, 5 µg/mL insulin, 10 µM pioglitazone) with or without 10 µM RU486 for 3 days, followed by treatment with 5 µg/mL insulin alone for 3 days. These treatments were repeated. Cells were stained with Oil Red O at 12 days post-induction (a). Quantitative real time PCR for the determination of expression levels adipogenic genes on day 2 after AIM exposure in the presence or absence of RU486 (b). MEFs were differentiated into adipocytes with AIM for 3 days, followed by treatment with insulin alone for 4 days. These treatments were repeated. Cells were stained with Oil Red O at 14 days post-induction. Macroscopic (upper panel) and microscopic (lower panel) views are shown (c). Data from 3 independent experiments are mean ± SEM; n=6. P values were determined by Steel-Dwass’ test (* P <0.05 vs control, # P<0.05 vs AIM).
Figure 2. Induction of Adipocyte Differentiation in MEFs from GRdim and GRnull Mice
MEFs derived from wild type, GRnull and GRdim mice were differentiated into adipocytes and stained with Nile Red for lipid droplets (yellow) and DAPI (blue) (a). Total fluorescent intensity of lipid droplets per cell were analyzed by Cellomics automated fluorescent microscopy (b). Data were mean ±SEM; n=5. P values were determined by Mann-Whitney U test (** P<0.01 vs control). Analysis of mRNA expression of adipogenesis-specific factors in wild type, GRdim, and GRnull MEFs during adipogenesis using quantitative RT-PCR 1 day after adipogenic differentiation (c). Data are mean ±SEM; n=3. P values were determined by Mann-Whitney U test (** P<0.01 vs control, * P<0.05 vs control).
Figure 3. Screening for GR-Regulated Adipogenic Induced Genes by Microarray Expression Analysis
Scheme of microarray data analysis showing genes with increased or decreased expression in each treatment group (a).Genes with signal intensities below 50 were eliminated and normalized, and then genes from cells incubated with AIM medium or Dex treatment that exhibited at least a twofold change relative to the control treatment were selected for analysis. Expression profiles for GR-regulated adipogenic genes in MEF were confirmed with quantitative RT-PCR in Control, AIM, AIM+RU, and Dex treatment groups on Day 2(b).Data are mean ±SEM; n=4. P values were determined by Steel-Dwass’ test (* P<0.05 vs control, # P<0.05 vs AIM).
Figure 4. Expressions of KLF15 and C/EBPδ mRNA in MEFs from Wild Type, GRdim and GRnull Mice
Expression of KLF15 and effect of RU486 for KLF15 in hMSCs 2days after induction (a) and expression of KLF15 and C/EBPδ in wild type, GRdim and GRnull MEFs during adipogenesis (b). Data are mean±SEM; n=3. Data were statistically analyzed with Steel’s test (* P<0.05 vs wild type 0hr, # P<0.05 vs GRnull 0hr, †P<0.05 vs GRdim 0hr). Time courses of expression of KLF15 and adipogenic marker genes in hMSCs(c). Expressions of KLF15 and C/EBPδ in wild type MEFs at 6 hours after Dex alone stimulation(d). Total RNA was analyzed using quantitative RT-PCR. Data are mean±SEM; n=3–4. P values were determined by Mann-Whitney U test (* P<0.05 vs control).
Figure 5. GRE Sites in the KLF15 Gene First Intron
The highly conserved region between human and mouse in the KLF15 first intron (a). GRE site sequence in the first intron of KLF15 (b). The conserved region between human and mouse genomes was identified by rVista 2.0 and the homology graph from the ECR browser, and putative GRE sites were discovered with MatInspector. Chromatin immunoprecipitation was performed in MEFs differentiated with AIM for 2 days. DNA immunoprecipitated with anti-GR antibodies from treated cells was used to amplify the nucleotide sequences located between +933 to +1070 bp (GRE1), and +1055 to +1216 bp (GRE2) in the KLF15 first intron(c). I: input, IgG: normal rabbit serum IgG.
Figure 6. Promoter Activity of Reporter Constructs Containing +933bp to +1216bp of the KLF15 First Intron
Map of pGL3 intKLF15 tk reporter vector (a). 293T cells were transiently transfected with the reporter construct pGL3-intKLF15-tk (b) or deletion constructs pGL3-GRE1mut (GRE1 mutation), pGL3-GRE2mut (GRE2 mutation), pGL3-GRE1&2mut (GRE1 and GRE2 mutations) (c), and empty vector (pcDNA) or GR expression vector. After 24 hr, cells were incubated in Dex (100 nM) or DMSO (vehicle) with or without 10 µM RU486 for 24 hr before the luciferase assay. Results were expressed as fold induction compared to cells treated with DMSO alone. Data are expressed as the mean ± SD of triplicates from representative experiments. Results were evaluated by Steel’s-test (* P< 0.05 vs pGL3-intKLF15-tk with Dex).
Figure 7. Effect of KLF15 Knockdown on Adipogenesis in MEFs
50nM siRNA for KLF15 (siKLF15) and 50nM nonspecific siRNA(siNega) were transfected into MEFs. After 48hr, cells were differentiated into adipocytes in the same way as wild type MEFs Figure 1 and were stained with Oil Red O at 14 days post-induction (a). Expression of KLF15 and adipogenic-specific marker genes in siRNA-transfected MEFs(b). Total RNA was extracted from cells 2 days after initiating adipose differentiation and analyzed using quantitative RT-PCR. Data are mean ±SEM; n=6–10. P values were determined by Mann-Whitney U test (** P<0.01 vs siNega, * P<0.05 vs siNega).
Figure 8. Schematic Model of the Pathways Controlling Differentiation
GR directly regulates KLF15 expression, which acts as an adipocyte differentiation activator at the transcriptional level in hMSCs and MEFs.
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