Liver-enriched inhibitory protein (LIP) actively inhibits preadipocyte differentiation through histone deacetylase 1 (HDAC1)

Abstract

The CCAAT/enhancer-binding protein β (C/EBPβ) is expressed as three isoforms (LAP*, liver-enriched activating protein (LAP), and liver-enriched inhibitory protein (LIP)) that differentially regulate gene expression. The interplay between LAP*, LAP, and LIP in regulating cellular processes is largely unknown, and LIP has been largely regarded to repress transcription through a passive heterodimerization-dependent mechanism. Recently, we have shown that p300/GCN5 and mSin3A/HDAC1 differentially regulate the ability of C/EBPβ to stimulate preadipocyte differentiation through activation of C/ebpα transcription. Here, we have mapped requirements for binding of mSin3A/HDAC1 to LAP/LAP* and LIP to a 4-amino acid motif in the central region of LAP/LAP* (residues 153-156) and the N terminus of LIP. Reducing mSin3A/HDAC1 binding to LAP/LAP* and LIP through deletion of this motif reduced the recruitment of HDAC1 to the C/ebpα promoter and increased preadipocyte differentiation stimulated by insulin and 1-methyl-3-isobutylxanthine. Additional studies showed that the interaction of HDAC1 with LIP provides for active repression of C/ebpα transcription and is largely responsible for the ability of LIP and HDAC1 to repress preadipocyte differentiation. Thus, although mSin3A/HDAC1 interacted readily with LAP/LAP* in addition to LIP and that expression of LAP/LAP* was sufficient to recruit HDAC1 to the C/ebpα promoter, mutations in C/ebpβ that abrogated HDAC1 association to LAP/LAP* in the absence of LIP provided no additional stimulation of differentiation or transcription beyond the deletion of LIP alone. The implication of these results for the interaction between p300/GCN5 and mSin3A/HDAC1 in regulating C/EBPα transcription and preadipocyte differentiation are discussed.

FIGURE 1.

Amino acids 153–156 and the bZip domain of C/EBPβ are required for mSin3A binding. A, schematic presentation of the three isoforms of the C/EBPβ. The schema shows the positions of N-terminal activation domains of LAP*/LAP and the bZip domain common to the three isoforms that mediates homo/heterodimerization and DNA binding. GCN5-dependent acetylation of C/EBPβ has been mapped to a cluster of lysines 98, 101, and 102 that occur in LAP*/LAP (Ac). Additional regulatory domains (RD1 and RD2) proposed by William et al. (30) are also indicated. B, quantification of radioblots of the binding of ³⁵S-labeled in vitro translated mSin3A to a series of GST-C/EBPβ fusion peptides with N-terminal (108C) or C-terminal (N107 to N217) truncations (n = 3, ± S.D., * = p < 0.05). C, ³⁵S-mSin3A binding to GST-C/EBPβ with internal deletions of amino acids 151–156 and 141–168 (C/EBPβ_Δ151–156 and C/EBPβ_Δ141–168) as in A, with quantification indicated below the radioblot (top) (n = 3, ± S.D., p < 0.01). The lower panel shows a Coomassie stain of SDS-PAGE separation of the input GST fusion peptides. D, COS7 cells were cotransfected with expression plasmids for FLAG-HDAC1 and either C/EBPβ_wt or C/EBPβ_Δ153–156. Whole cell lysates were immunoprecipitated with a FLAG affinity resin, with 10% of input material being shown on the right. Coimmunoprecipitation of LAP*/LAP with FLAG-HDAC1 was quantified as shown (n = 3, ± S.D., p < 0.01). E, Western analysis showing the expression level of exogenous C/EBPβ constructs expressed by transient transfection in NIH 3T3 cells. IP, immunoprecipitation. F, transient transfection analysis of the transcriptional activation potential of C/EBPβ_wt and C/EBPβ_Δ153–156 at the C/epbα promoter (−350/+7) measured from a luciferase reporter gene. 100 ng of HDAC1 expression vector was also transfected as indicated. Luciferase activity was corrected for transfection efficiency by using a cotransfected Renilla expression plasmid, and values are represented as relative luciferase unit (RLU) (n = 4 duplicates, ± S.E., ** = p < 0.02).

FIGURE 2.

C/EBPβ_Δ153–156 enhances differentiation of NIH 3T3 cells to mature adipocytes. A, NIH 3T3 cells retrovirally transduced to express either C/EBPβ_wt or C/EBPβ_Δ153–156 as described previously (6) were harvested 4 days following treatment with insulin and MIX in the absence (MI) or presence of 250 nm dex (MID), and whole cell extracts were analyzed by Western blot for adipsin and actin levels (upper panel). The cells in duplicate experiments were also stained with Oil Red O 6 days after treatment to reveal lipid accumulation (2× magnification, lower panel). B, Western analysis of stably expressed C/EBPβ_wt or C/EBPβ_Δ153–156 in NIH 3T3 cells on the day of initiation of treatment (day 0). C, to assess the presence of HDAC1 and C/EBPβ on the C/epbα promoter, C/EBPβ expressing NIH 3T3 cells were treated with MI for 24 h prior to ChIP analysis using HDAC1 and C/EBPβ antibodies as indicated. The PCR-amplified DNA fragment shown shows the binding of C/EBPβ or HDAC1 to region −460/−244 of the C/epbα promoter. The figure is representative of the results of three independent experiments. D, C/epbα (top) and Pparγ2 (bottom) mRNA expression was determined by real time RT-PCR 24 and 48 h following treatment of NIH 3T3 cells with MI or MID. Values were normalized to actin (n = 3, ± S.D., * = p < 0.05). C/epbα quantification is relative to the MI pLXSN control (set as 1 relative unit (RU)), whereas Pparγ2 values are relative to the MID β_wt condition and again defined as 1 relative unit.

FIGURE 3.

C/EBPβ_Δ153–156 expression enhances differentiation of 3T3 L1 preadipocytes. A, Western blot analysis of stably expressed LAP, LAP*, and LIP levels expressed from C/EBPβ_wt or C/EBPβ_Δ153–156 retroviral vectors in 3T3 L1 cells 2 days following confluency. B and C, differentiation of retrovirally transduced 3T3 L1 cells expressing control (pLXSN) or C/EBPβ_wt and C/EBPβ_Δ153–156 constructs and cultured in the absence of insulin, MIX, or dex. Western blot analysis of the expression of adipsin, C/EBPα, PPARγ, actin (B) and photomicrograph of Oil Red O staining of accumulated lipid (4× magnification) (C) was performed at day 8 of differentiation.

FIGURE 4.

Mutation reducing LIP association with HDAC1 compromises LIP-mediated inhibition of C/epbα expression in the absence of dex. A, coimmunoprecipitation assay with extracts of 3T3 L1 cells stably expressing WT LIP or LIP_6C via retroviral transduction. Binding is compared with 5% of input, and levels of nonspecific (NS) binding were assessed using an antibody against Gal4 DNA-binding domain. Quantification of relative binding to HDAC1 is at the bottom of the panel and reflects three independent experiments (± S.D., p < 0.05). B, analysis of C/EBPα promoter (−350/+7) activity in NIH 3T3 cells by a luciferase assay following transient transfection with 50 ng of WT C/EBPβ expression vector together with the indicated amounts of LIP or LIP_6C expression vectors. Cells were treated with vehicle (−DEX, top panel) or 1 μm dex (+DEX, bottom panel) for 16 h following transfection. RLU, relative luciferase unit. The results shown are quantified from three independent experiments performed in duplicate (± S.E., * = p < 0.05). C, Western analysis showing the expression level of transiently transfected C/EBPβ alone or cotransfected with 100 ng of either LIP or LIP_6C in NIH 3T3 cells treated with either vehicle (ethanol) or dex.

FIGURE 5.

Reducing mSin3A/HDAC1 binding compromises the ability of LIP to repress preadipocyte differentiation. A, 3T3 L1 cells retrovirally transduced to express either pLXSN vector, LIP_wt, or LIP_6C were differentiated for 8 days following MID treatment. A Western blot analysis was performed to assess adipsin, C/EBPα, PPARγ, and actin levels. The reduced level of actin expression in the pLXSN lanes reflects a 3-fold decrease in the amount of extract used, which was required to prevent overexposure of the pLSXN adipsin track on the blot. B, time course Western analysis of endogenous C/EBPβ levels in 3T3 L1 cells following their treatment with either MI (−) or MID (+) at 2 days post-confluency. C, Western blot analysis showing the expression level of endogenous (pcDNA mock plasmid) and exogenous LIP in 3T3 L1 cells 3 days following transfection with 100 ng of the DNA constructs (left panel). The transfected 3T3 L1 cells were also harvested 2 days post-treatment (or 5 days following transfection with the LIP constructs) to evidence a decrease in exogenous LIP expression, as shown by similar expression levels in all three lanes (right panel). D, 1 day before confluency, 3T3 L1 cells were transiently transfected with increasing amount of the pcDNA-LIP or pcDNA-LIP_6C constructs. Three days later, the cells were induced to differentiate with MID for 8 days and harvested for immunoblot analysis as in A, except in this instance loading was equal in all lanes.

FIGURE 6.

Repressive effect of mSin3A/HDAC1 association with C/EBPβ on C/ebpα expression is mediated primarily through LIP association. A, Western analysis of the expression levels of exogenous WT C/EBPβ, C/EBPβ_M152A, and C/EBPβ_Δ151–156 in NIH 3T3 cells expressed by a transient transfection. B, coimmunoprecipitation analysis of the binding of FLAG-HDAC1 to C/EBPβ_wt, C/EBPβ_M152A, and C/EBPβ_Δ151–156. Whole cell lysates prepared from transiently transfected COS7 cells were immunoprecipitated (IP) with a FLAG affinity resin. C, chromatin immunoprecipitation analysis of HDAC1 association with the C/epbα promoter in NIH 3T3 cells stably expressing retroviral C/EBPβ constructs. Cells were treated with MI for 24 h prior to analysis. A representative result from three independent experiments is shown. D, transient transfection analysis of the activation of C/epbα-luciferase expression by C/EBPβ, C/EBPβ_M152A, and C/EBPβ_Δ151–156 in NIH 3T3 cells. Cells were treated with either vehicle (□) or 1 μm dex (■) for 16 h following transfection. Numbers represent the averages of four independent experiments performed in duplicate (± S.E., * = p < 0.05).

FIGURE 7.

Repressive effect of mSin3A/HDAC1 association with C/EBPβ on preadipocyte differentiation is mediated primarily through LIP association. A, Western blot analysis of stably expressed C/EBPβ_wt, C/EBPβ_M152A, and C/EBPβ_Δ151–156 in NIH 3T3 cells just prior to the initiation of differentiation (day 0). B, Western analysis of adipsin and actin in cells harvested 4 days post-induction in the absence (MI) or presence (MID) of 250 nm dex. C, real time RT-PCR analysis of C/epbα (upper panel) and Pparγ2 (lower panel) mRNA expression at 24 h following the induction of differentiation with MI (□) or MID (■). mRNA levels were normalized to actin (n = 3, ± S.D., * = p < 0.05 and ** = p < 0.01). Quantification of both C/EBPα and PPARγ2 are relative to MID β_wt, set as 1 relative unit.

FIGURE 8.

Schematic presentation of the interplay of p300/GCN5 and mSin3A/HDAC1 interacting with C/EBPβ at the C/EBPα promoter. Molecular complex assembly nucleated by C/EBPβ isoforms and dimer combinations at the C/EBPα promoter in preadipocytes stimulated to differentiate with insulin and MIX in the absence (left) or presence of dex treatment (right). LAP/LAP* (represented simply here as LAP), LIP, p300/GCN5, mSin3A, and HDAC1 are represented as indicated. Ac = acetylation at amino acids 98–102 in LAP/LAP* that interferes with mSin3A binding to C/EBPβ (6). The relative levels of transcription are reflected by the sizes of arrows. The dotted line representing HDAC1 in the lower right panel reflects the decrease in HDAC1 levels that occurs in response to dex treatment, which would be expected to reduce the recruitment of HDAC1 to the promoter.