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. 2003 May 1;22(9):2135-45.
doi: 10.1093/emboj/cdg218.

Stimulation of preadipocyte differentiation by steroid through targeting of an HDAC1 complex

Nadine Wiper-Bergeron  1 , Dongmei Wu, Louise Pope, Caroline Schild-Poulter, Robert J G Haché
Affiliations

Stimulation of preadipocyte differentiation by steroid through targeting of an HDAC1 complex

Nadine Wiper-Bergeron et al. EMBO J. .

Abstract

Glucocorticoids potentiate the early steps of preadipocyte differentiation and promote obesity in Cushing's syndrome and during prolonged steroid therapy. We show that glucocorticoids stimulate 3T3 L1 preadipocyte differentiation through a non-transcriptional mechanism mediated through the ligand-binding domain of the glucocorticoid receptor. This enhanced the onset of CCAAT/enhancer binding protein (C/EBPalpha) expression by potentiating its initial transcriptional activation by C/EBPbeta. In the absence of steroid, C/EBPbeta associated with a transcriptional corepressor complex containing mSin3A and histone deacetylase 1 (HDAC1), but lacking HDAC2 and RbAp46/48. HDAC1/mSin3A were recruited to the C/EBPalpha promoter with C/EBPbeta and promoted the deacetylation of histone H4. Steroid induced the specific depletion of this corepressor by targeting the HDAC1 within the complex for degradation through the 26S proteasome. Treatment with histone deacetylase inhibitors replaced the effects of steroid treatment on preadipocyte differentiation and C/EBPalpha expression, while overexpression of HDAC1 abrogated the stimulatory effects of steroid. Recapitulation of the glucocorticoid effect by progestin treatment in the presence of the progesterone receptor ligand-binding domain suggests a conserved mechanism relevant to many aspects of steroid-mediated differentiation.

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Figures

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Fig. 1. Enhancement of preadipocyte differentiation is mediated through steroid receptor LBDs. (A) Oil red O staining, adipsin protein levels and GDPH activity of 3T3 L1 cells infected with retrovirus to express amino acids 505–795 of rat GR (GR505C) or control virus (pLXSN) and cultured for 8 days in the presence of MIX and 50 nM insulin (+MI), MIX, insulin and dex (+MID), or in the absence of cocktail (–C). The images displayed are representative of results observed in a minimum of three independent experiments performed in duplicate over a period of several months. For GPDH, 1 mU equals 1 nmol of product/min/mg of protein. (B) Effect of expression of amino acids 632–933 of human PR (PR632C) on 3T3 L1 differentiation as visualized by Oil red O staining. (C) Western analysis of C/EBPβ, δ, α and PPARγ expression in 3T3 L1 cells following 24 h treatment with MIX, insulin and dex (D) or R5020 (R).
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Fig. 2. GR and PR LBDs potentiate C/EBPβ-dependent C/EBPα expression. (A) Fold induction of luciferase activity from the C/EBPα (–355/+7) and PPARγ (–609/+52) promoters upon ectopic expression of C/EBPα, β and δ in HeLa cells. (B) Fold increase of C/EBPα, β and δ-dependent luciferase activity by dex treatment above the level of activity in (A). (C) Effect of RU486 on the potentiation of C/EBPβ-dependent luciferase activity from the C/EBPα promoter in HeLa cells. (D) Fold increase of C/EBPβ-dependent luciferase activity from the C/EBPα promoter upon dex treatment of Cos7 cells cotransfected with GR, GRL501P or GR505C. (E) Mutation of C/EBP binding site in the C/EBPα promoter abrogates the GR505C-dependent stimulation of C/EBPα transcription in Cos7 cells. The schema delimits the 29 bp mutation that abrogrates the C/EBP response element. (F) Comparison of the potentiation of C/EBPβ-dependent luciferase activity from the C/EBPα promoter by GR505C, PR632C and RARα upon treatment with dex, R5020 and retinoic acid.
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Fig. 3. Interaction of HDAC1 with C/EBPβ is relieved by prolonged steroid treatment. (A) Effect of transiently transfected C/EBPβ, GR505C, PR632C and HDAC1 on the induction of luciferase activity from the C/EBPα promoter in Cos7 cells. (B) Effect of dex and TSA on the fold induction of C/EBPβ-dependent luciferase activity from the C/EBPα promoter in Cos7 cells. (C) Immunoprecipitation of transient transfected C/EBPβ (β) and endogenous HDAC1(H) from Cos7 cells (top) or endogenous C/EBPβ and HDAC1 from 3T3 L1 cells treated for 24 h with MIX and 100 nM insulin (bottom). NS, non-specific type-matched antibody. Immunoprecipitates and 10% of the extracts used for immunoprecipitation (input) were resolved by SDS–PAGE and probed for the presence of the indicated proteins with specific antibodies. (D) GST pull-down assay of the binding of in vitro translated mSin3A and HDAC1 to GSTC/EBPβ and GST. (E) Immunoprecipitations from 3T3 L1 cells performed as described in (C) except that dex was included for the final 4 h or full 24 h of insulin/MIX treatment as indicated. (F) Northern analysis of HDAC1 mRNA following insulin, MIX and dex treatment compared with 18S rRNA.
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Fig. 4. Histone deacetylases repress preadipocyte differentiation. (A) C/EBPα, β and δ levels at 24 h in whole-cell extracts prepared from 3T3 L1 cells treated with insulin and MIX and dex as indicated, and infected with pLXSN or HDAC1-expressing virus (left) or treated with TSA (400 nM) or valproic acid (VPA; 10 mM, right). (B) Effect of initial 48 h TSA or VPA treatment on Oil red O staining and adipsin expression at day 8 in 3T3 L1 cells stimulated with insulin and MIX (+MI) and dex (+DEX). (C) Effect of virally mediated HDAC1 expression on differentiation of 3T3 L1 cells as revealed by adipsin levels and Oil red O.
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Fig. 5. Dex induces selective loss of an HDAC1/mSin3A complex that preferentially interacts with C/EBPβ. (A) Immunoprecipitates from 3T3 L1 cells (top) and Cos7 cells transfected to express the GR LBD (bottom) were prepared as described in Figure 3C except using an antibody to GR (IP GR) and including dex treatment for the final 4 h of the insulin/MIX (MI) treatment. Western analysis of the immunoprecipitates was performed with antibodies to the factors indicated (B) Effect of MG132 (1 µM) on levels of HDAC1, mSin3A and GR in 3T3 L1 cells treated with insulin/MIX and dex for 24 h (top). Effect of 24 h R5020 and MG132 on endogenous HDAC1 levels in Cos7 cells transfected with PR632C (middle) or effect of 24 h dex/MG132 treatment on endogenous and transiently expressed (HDAC1-HA) HDAC1 in Cos7 cells transfected with GR505C. (C) Quantitative display of western analysis of HDAC1 levels in FPLC fractions prepared from 3T3 L1 cells treated with for 24 h with insulin/MIX (dark line) or insulin/MIX/dex (gray line). Curves are standardized against total HDAC1 levels in extracts from untreated cells. Peaks are labelled I–VI, with IVa indicating a shift in the position of peak IV upon dex treatment (D) Quantification of HDAC1, mSin3A, RbAp48 and C/EBPβ levels in fractions from (C) for extract prepared from cells treated with insulin/MIX only. Calculations of percent HDAC1, mSin3A, RbAp48 and C/EBPβ represent areas under the curve for each peak.
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Fig. 6. MG132 abrogates the effect of dex on recruitment of HDAC1/mSin3A to the C/EBPα promoter. (A) ChIP analysis of the C/EBPα promoter in 3T3 L1 cells treated for 24 h with vehicle (–C), insulin (I), MIX (M) and dex (D) as indicated. Formaldehyde-crosslinked DNA–protein complexes were immunoprecipitated with the antibodies to the proteins indicated and with a pan-acetyl histone H4 antibody (AcH4). DNAs prepared from the extracts employed for the immunoprecipitations were used as input controls (B) ChIP for AcH4 in confluent 3T3 L1, SF7 and NIH 3T3 cells. (C) Effect of MG132 treatment (1 µM) on the dex-dependent fold increase of C/EBPβ-dependent luciferase activity from the C/EBPα promoter in Cos7 cells cotransfected with GR505C. (D) Effect of MG132 treatment (1 µM) on ChIP for HDAC1 on C/EBPα promoter in 3T3 L1 cells performed as in (A).
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