NCoR1 regulates thyroid hormone receptor isoform-dependent adipogenesis - PubMed (original) (raw)
NCoR1 regulates thyroid hormone receptor isoform-dependent adipogenesis
Xu-Guang Zhu et al. J Mol Endocrinol. 2011.
Abstract
We previously showed that two thyroid hormone receptor (TR) isoforms--TRα1 and TRβ1--differentially regulate thyroid hormone (triiodothyroxine, T(3))-stimulated adipogenesis in vivo. This study aims to understand the role of the nuclear receptor corepressor, NCoR1, in TR isoform-dependent adipogenesis. We found that T(3)-stimulated adipogenesis of 3T3-L1 cells was accompanied by progressive loss of NCoR1 protein levels. In 3T3-L1 cells stably expressing a mutated TRα1, PV (L1-α1PV cells), the T(3)-stimulated adipogenesis was more strongly inhibited than that in 3T3-L1 cells stably expressing an identical mutation in TRβ1 (L1-β1PV cells). The stronger inhibition of adipogenesis in L1-α1PV cells was associated with a higher NCoR1 protein level. These results indicate that the degree of loss of NCoR1 correlates with the extent of adipogenesis. siRNA knockdown of NCoR1 promoted adipogenesis of control 3T3-L1 cells and reversed the inhibited adipogenesis of L1-α1PV and L1-β1PV cells, indicating that NCoR1 plays an essential role in TR isoform-dependent adipogenesis. An ubiquitin ligase, mSiah2, that targets NCoR1 for proteasome degradation was upregulated on day 1 before the onset of progressive loss of NCoR1. NCoR1 was found to associate with mSiah2 and with TR, TRα1PV, or TRβ1PV, but a stronger interaction of NCoR1 with TRα1PV than with TRβ1PV was detected. Furthermore, TRα1PV-NCoR1 complex was more avidly recruited than TRβ1PV-NCoR1 to the promoter of the C/ebpα gene, leading to more inhibition in its expression. These results indicate that differential interaction of NCoR1 with TR isoforms accounted for the TR isoform-dependent regulation of adipogenesis and that aberrant interaction of NCoR1 with TR could underlie the pathogenesis of lipid disorders in hypothyroidism.
Conflict of interest statement
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Figures
Figure 1
Abundance of NCoR1 protein in control cells, L1-β1PV cells, and L1-α1PV cells on day 1 (A-a), day 2 (A-b), and day 6 (A-c) after differentiation induction in the absence of T3 or in the presence of T3 (2 nM). (A) Representative results of western blot analysis. Cellular extracts were prepared from adipocytes, and western blot analysis was carried out using anti-NCoR1 #1975/1976 antibody as described in ‘Materials and methods’. The experiments were performed with five replicates in each cell line. GAPDH was used as a loading control. (B) Quantitative analysis for the relative abundance of NCoR1 protein on day 6 (A-c) compared with that on day 1 (A-a) in the absence of T3 (bars 1–3) or in the presence of T3 (bars 4–6). NCoR1 protein level was normalized against the GAPDH protein level. The P values are indicated. NS, not significant.
Figure 2
Abundance of NCoR1 protein in control cells, L1-β1PV cells, and L1-α1PV cells after transduction with scrambled-siRNA or NCoR1-siRNA lentiviruses. (A) Representative results of western blot analysis. Cellular extracts were prepared and western blot analysis was carried out using anti-NCoR1 #1975/1976 antibody (upper panel) and anti-GAPDH antibody (lower panel) as loading control. The experiments were performed with three replicates for each cell line. (B) Quantitative analysis for the relative abundance of NCoR1 protein. NCoR1 protein levels were normalized against the GAPDH protein level. The P values are indicated. NS, not significant.
Figure 3
Knocking down of NCoR1 expression by NCoR1-specific siRNA promotes adipogenesis in control cells, L1-β1PV cells, and L1-α1PV cells. Cells treated with scrambled siRNA (plates 1–3 of A and panels a–c of B) or NCoR1-specific siRNA (plates 4–6 of A and panels d–f of B) were induced to differentiate into mature adipocytes in the presence of T3 (2 nM). The experiments were performed with three replicates for each cell line. Mature adipocytes with lipid droplets were visualized by staining with Oil Red-O (A) and by phase contrast microscopy (B). (C) Quantitative analysis for the relative intensities of Oil Red-O staining in control cells (bars 1–2), L1-β1PV cells (bars 3–4), and L1-α1PV cells (bars 5–6). The P values are indicated.
Figure 4
Expression of NCoR1 mRNA in control cells, L1-β1PV cells, and L1-α1PV cells on days 1, 2, and 6 after induction of adipogenesis. Total RNAs were prepared from control, L1-β1PV, and L1-α1PV cells in the absence of T3 or in the presence of T3 (2 nM) after induction of adipogenesis. An aliquot of RNAs (200 ng) was used in quantitative real-time PCR reaction with mouse-specific NCoR1 primers or 18S primers (control primer). Relative NCoR1 mRNA expression was normalized against 18S rRNA level. The experiments were performed with three replicates for each cell line. The P values are indicated.
Figure 5
Interaction of TRs and NCoR1 in control cells, L1-β1PV cells, and L1-α1PV cells on day 6 after induction of adipogenesis (A and B) or before induction of adipogenesis (C and D). The experiments were performed with three replicates for each cell line. (A) Co-immunoprecipitation (Co-IP) of TRα1PV, TRβ1PV, and wild-type TRβ1 with NCoR1 on day 6 after induction of adipogenesis was performed with antibody against mutant PV at the carboxyl terminus (#302) (lanes 4–6, panel A-a), antibody against wild-type TR (C4) (lanes 2–3, panel A-b), or IgG (lanes 7–9, panel A-a and lanes 4–5 of panel A-b). Western blot analysiswascarried out using anti-NCoR1 #1975/1976 antibody. For input controls, 20% of cell lysate were used. (B) Quantitative analysis for the relative abundance of NCoR1 protein in L1-β1PV cells and L1-α1PV cells after Co-IP with TRβ1PV and TRα1PV as shown in panel A-a (lanes 5–6). (C) Co-IP of TRα1PV, TRβ1PV, and wild-typeTRβ1 with NCoR1 before induction of adipogenesis was performed with antibody against mutant PV at the carboxyl terminus (#302) (lanes 4–6, panel C-a), antibody against wild-type TR (C4) (lanes 2–3, panel C-b), or IgG (lanes 7–9 of panel C-a and lanes 4–5 of panel C-b). Western blot analysis was carried out using anti-NCoR1 #1975/1976 antibody. For input control, 5% of cell lysates were used. (D) Quantitative analysis for the relative abundance of NCoR1 protein in L1-β1PV cells and L1-α1PV cells after Co-IP with TRβ1PV and TRα1PV as shown in panel C-a (lanes 5–6).
Figure 6
Abundance of mSiah2 protein during adipogenesis. (A) Abundance of mSiah2 protein in control cells, L1-β1PV cells, and L1-α1PV cells without induction or on day 1 after induction of adipogenesis. Cellular extracts were prepared from adipocytes and western blot analysis was carried out using anti-mSiah2 antibody. GAPDH was used as loading controls. The experiments were performed with four replicates in each cell line. (B) Abundance of mSiah2 protein in control cells, L1-β1PV cells, and L1-α1PV cells on days 1, 2, and 6 after induction of adipogenesis in the absence of T3 (lanes 1, 3 and 5) or in the presence of T3 (lanes 2, 4 and 6). Cellular extracts were prepared from adipocytes and western blot analysis was carried out using anti-mSiah2 antibody. GAPDH was used as loading controls. The experiments were performed with three replicates for each cell line. (C) Quantitative comparison for the relative intensities of mSiah2 protein in control cells, L1-β1PV cells, and L1-α1PV cells without induction or after induction of adipogenesis as shown in panel A. (D) Quantitative comparison for the relative intensity of mSiah2 protein in control cells, L1-β1PV cells, and L1-α1PV cells in the presence of T3 versus in the absence of T3 as shown in panel B. *Indicates P<0·05.
Figure 7
Interaction of TR/NCoR1/mSiah2 proteins. (A) Interaction of mSiah2 with NCoR1 in control cells, L1-β1PV cells, and L1-α1PV cells before induction of adipogenesis. For input controls, 5% of cell lysate were used. Co-IP of mSiah2 with NCoR1 was performed with anti-NCoR1 #1975/1976 antibody (lanes 4–6) or IgG (control) (lanes 7–9). The immunoprecipitated proteins were analyzed by western blot analysis with antibody against mSiah2. (B). After Co-IP with antibody C4 (lane 5), #302 (lanes 4 and 6–7), or IgG (lanes 8–10), the immunoprecipitated proteins were analyzed by western blot analysis using antibody against mSiah2.
Figure 8
Preferential recruitment of NCoR1 by TRα1PV versus TRβ1PV to the promoter of the _C/ebp_α gene. (A) Chromosomal DNA was prepared from control, L1-β1PV, and L1-α1PV cells on day 6 after induction of adipogenesis and ChIP was carried out as described in ‘Materials and methods’. The chromosomal DNA was incubated with IgG (control) and anti-NCoR1 (PHQQ) antibody. The purified DNA from immunoprecipitated chromatin was used as a template for PCR amplification of the receptor-binding region in the promoter of _C/ebp_α gene (A, upper panel). For the input DNA, 1% of the chromatin solution (10 µl) was used as a control (A, lower panel). The experiments were performed with three replicates for each cell line. (B) DNA gel images were scanned and subjected to band densitometry analysis with imageJ software. Data are expressed as means±
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