Impaired adipogenesis caused by a mutated thyroid hormone alpha1 receptor - PubMed (original) (raw)

Impaired adipogenesis caused by a mutated thyroid hormone alpha1 receptor

Hao Ying et al. Mol Cell Biol. 2007 Mar.

Abstract

Thyroid hormone (T3) is critical for growth, differentiation, and maintenance of metabolic homeostasis. Mice with a knock-in mutation in the thyroid hormone receptor alpha gene (TRalpha1PV) were created previously to explore the roles of mutated TRalpha1 in vivo. TRalpha1PV is a dominant negative mutant with a frameshift mutation in the carboxyl-terminal 14 amino acids that results in the loss of T3 binding and transcription capacity. Homozygous knock-in TRalpha1(PV/PV) mice are embryonic lethal, and heterozygous TRalpha1(PV/+) mice display the striking phenotype of dwarfism. These mutant mice provide a valuable tool for identifying the defects that contribute to dwarfism. Here we show that white adipose tissue (WAT) mass was markedly reduced in TRalpha1(PV/+) mice. The expression of peroxisome proliferator-activated receptor gamma (PPARgamma), the key regulator of adipogenesis, was repressed at both mRNA and protein levels in WAT of TRalpha1(PV/+) mice. Moreover, TRalpha1PV acted to inhibit the transcription activity of PPARgamma by competition with PPARgamma for binding to PPARgamma response elements and for heterodimerization with the retinoid X receptors. The expression of TRalpha1PV blocked the T3-dependent adipogenesis of 3T3-L1 cells and repressed the expression of PPARgamma. Thus, mutations of TRalpha1 severely affect adipogenesis via cross talk with PPARgamma signaling. The present study suggests that defects in adipogenesis could contribute to the phenotypic manifestation of reduced body weight in TRalpha1(PV/+) mice.

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Figures

FIG. 1.

(A) Schematic representation of the structure of wild-type TRα1 and TRα1PV. The C-terminal, mutated sequence of TRα1PV is indicated. (B) Reduced WAT mass in TRα1PV/+ mice. Inguinal, epididymal, perirenal, and interscapular fat tissues were weighed after dissection from mice at ages of 3 to 6 months. Total fat represents the sum of these four isolated fat tissues. Ratios of fat mass versus body weight were determined and are shown in panel a. Mouse body weights are shown in panel b. The data are expressed as means ± SEM (error bars) (n = 15 to 16). (C) Increased food consumption in TRα1PV/+ mice. Food consumed by mice in 2 days was measured, and the food intake was normalized to the body weight of each mouse and expressed as g/g body weight−0.75 (24, 50). Each circle represents an individual mouse, and the horizontal bars represent the mean values. The differences are statistically significant (P < 0.0001). WT, wild type; N.S., no significant difference.

FIG. 2.

Expression of TRs in 3T3-L1 cells (A) and WAT of wild-type mice (B). (A) Cellular extracts were prepared from 3T3-L1 preadipocytes (lane 1) and mature adipoctyes (lane 2), and Western blot analysis was carried out using monoclonal anti-TR antibody C4 that recognized TRα1 and TRβ1 as described in Materials and Methods. Only TRα1 was detected. (B) WAT of wild-type mice was fractionated into mature adipocytes and stromal vascular fraction enriched with preadipocytes as described in Materials and Methods. A QuantiTect SYBR green RT-PCR kit (QIAGEN) and LightCycler software were used for the absolute quantification of TRα1 and TRβ1 mRNA levels in mature adipocytes and stromal vascular fraction, respectively. mRNA abundance of TRα1 and TRβ1 was normalized by 18S and shown as mRNA expression relative to the expression of TRα1 in the stromal vascular fraction, defined as 1. Error bars indicate standard errors.

FIG. 3.

Comparison of in vitro norepinephrine-mediated lipolysis in isolated white fat cells. White fat cells were isolated from the epididymal pad, and the amount of glycerol released in the presence of increasing concentrations of norepinephrine was determined as described in Materials and Methods. Closed circles and open circles represent data from TRα1PV/+ mice and wild-type mice, respectively. Data are expressed as means ± SEM (error bars) (n = 9).

FIG. 4.

Impaired adipogenesis in WAT of TRα1PV/+ mice. (A) There was a reduced number of mature adipocytes in TRα1PV/+ mice compared with that in wild-type (WT) mice. Mature adipocytes were separated from the stromal vascular fraction enriched with preadipocytes as described in Materials and Methods. (B) Reduced expression of key lipogenic enzymes in TRα1PV/+ mice. Total RNAs were prepared from mice aged 4 to 5 months, and quantitative real-time RT-PCR was performed using 0.1 μg of total RNA as described in Materials and Methods. Relative changes (_n_-fold) of the expression of mRNA are shown. The differences are significant (P < 0.05). The activity of G6PD was reduced in TRα1PV/+ mice compared with that in wild-type mice (C). The enzyme activity was determined as described in Materials and Methods. The differences are significant (P < 0.05). ΔOD, change in optical density. Error bars indicate standard errors.

FIG. 5.

Comparison of the expression levels of PPARγ and its downstream target genes at the mRNA levels in TRα1PV/+ mice and wild-type (WT) siblings (A) and PPARγ protein abundance (B). (A) mRNA expression of PPARγ and its downstream target genes in the WAT of TRα1PV/+ mice and wild-type siblings. Total RNAs were prepared from mice aged 4 to 5 months, and quantitative real-time RT-PCR was performed using 0.1 μg of total RNA as described in Materials and Methods. Relative changes (_n_-fold) of the expression of mRNA are shown. The differences are significant (P < 0.05). (B) Panel a represents nuclear extracts (25 μg) isolated from WAT (inguinal fat) of TRα1PV/+ mice (n = 2) and wild-type siblings (n = 3). Western blot analysis was carried as described in Materials and Methods. The primary antibody was the monoclonal anti-PPARγ antibody (Santa Cruz; catalog no. sc-7273; 1:100 dilution). Panel b shows the quantification of band intensities from panel a, and relative differences were plotted. The differences are significant (P < 0.001). For panel c, PARP was used for the control of protein loading (anti-PARP antibody [Santa Cruz; catalog no. sc-7150]; 1:100 dilution). Error bars indicate standard errors.

FIG. 6.

Effect of T3 on the mRNA expression of PPARγ in WAT of wild-type mice. Hypothyroid mice were induced by PTU treatment, and hyperthyroid mice were induced from injection of T3 as described in Materials and Methods. mRNA expression was determined by real-time RT-PCR. The data are expressed as means ± SEM (error bars) (n = 4 to 5).

FIG. 7.

TRα1PV represses the ligand-dependent transactivation of PPARγ in CV-1 cells. CV-1 cells were cotransfected with 0.5 μg of the reporter plasmid (pPPRE-TK-Luc), 0.1 μg of PPARγ expression vector (pSG5-mPPARγ 1), and 0.1 μg of TRα1 or TRα1PV expression vector (pcDNA3.1-TRα1 or pcDNA3.1-TRα1PV, respectively). Cells were treated with either dimethyl sulfoxide as vehicle or troglitazone (20 μM) in the absence (−) or presence (+) of T3 (100 nM), as marked. Data were normalized against the protein concentration in the lysates. Relative luciferase activity was calculated and shown as induction relative to the luciferase activity of PPRE in the cells treated with dimethyl sulfoxide in the absence of T3, defined as 1. The data are expressed as means ± SEM (error bars) (n = 3).

FIG. 8.

Binding of PPARα, TRα1, or TRα1PV to PPRE by EMSA. Lysate containing in vitro-translated PPARγ, TRα1, or TRα1PV proteins (5 μl) in the presence (+) or absence (−) of RXRβ or anti-TRβ1 antibody (C4; 2 μg), anti-PV antibody (T1; 2 μg), or an irrelevant antibody, MOPC (2 μg); antibodies were incubated with 32P-labeled PPRE and analyzed by gel retardation as described in Materials and Methods. Amounts of lysate were kept constant by the addition of unprogrammed lysate as needed.

FIG. 9.

Inhibition of the PPARγ/RXR heterodimer binding to PPRE by increasing amounts of TRα1 or TRα1PV. (A) Lysates containing PPARγ and RXRβ proteins (1 μl) were incubated in the absence (+) or presence (−) of TRα1 (1, 5, and 10 μl for lanes 4, 5, and 6, respectively) or TRα1PV (1, 5, and 10 μl for lanes 7, 8, and 9, respectively) with 32P-labeled PPRE and analyzed by EMSA as described in Materials and Methods. (B) Band intensities of lanes 2 and 4 through 9 from panel A were quantified by using UMAX Astra 6450 (UMAX Technologies, Inc., Dallas, TX), and the data were analyzed by using NIH Image 1.61. The data are expressed as means ± SEM (error bars) (n = 3). The asterisks indicate that the differences are significant (P < 0.01).

FIG. 10.

Recruitment of NCoR to PPRE-bound PPARγ and TRα1PV in the promoter of the lipoprotein lipase gene in WAT of TRα1PV/+ mice by ChIP assay. Nuclear extracts from wild-type (W) or TRα1PV/+ (M) mice were processed for the ChIP assay as described in Materials and Methods. Anti-PPARγ, anti-NCoR, anti-PV (polyclonal antibody T1 or monoclonal antibody 302) (4, 49) antibodies and IgG (for negative controls) were used for immunoprecipitation. The precipitated DNA was amplified by PCR with primers specific for PPRE in the lipoprotein lipase promoter, and the products were analyzed. −, no antibody.

FIG. 11.

TRα1PV impairs T3-dependent 3T3-L1 adipogenesis. 3T3-L1 cells were transfected with 6 μg of the expression vector for TRα1PV (FLAG-TRα1PV) or pFLAG-CMV2 as a control, and the transfected cells were induced to differentiate into mature adipocytes as indicated in Materials and Methods. Mature adipocytes with lipid droplets were visualized by staining with Oil Red-O (A) and shown by phase-contrast microscopy (B). Western blot analysis of PPARγ (C), TRα1PV/+ (D, panel a), and TRα1 (D, panel b) protein abundance was carried out as described in Materials and Methods. −, absence of; +, presence of.

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Impaired adipogenesis caused by a mutated thyroid hormone alpha1 receptor - PubMed (original) (raw)