Nuclear corepressors mediate the repression of phospholipase A2 group IIa gene transcription by thyroid hormone

Abstract

Secretory phospholipase A2 group IIa (PLA2g2a) is associated with inflammation, hyperlipidemia, and atherogenesis. Transcription of the PLA2g2a gene is induced by multiple cytokines. Here, we report the surprising observation that thyroid hormone (T3) inhibited PLA2g2a gene expression in human and rat hepatocytes as well as in rat liver. Moreover, T3 reduced the cytokine-mediated induction of PLA2g2a, suggesting that the thyroid status may modulate aspects of the inflammatory response. In an effort to dissect the mechanism of repression by T3, we cloned the PLA2g2a gene and identified a negative T3 response element in the promoter. This T3 receptor (TRβ)-binding site differed considerably from consensus T3 stimulatory elements. Using in vitro and in vivo binding assays, we found that TRβ bound directly to the PLA2g2a promoter as a heterodimer with the retinoid X receptor. Knockdown of nuclear corepressor or silencing mediator for retinoid and thyroid receptors by siRNA blocked the T3 inhibition of PLA2g2a. Using chromatin immunoprecipitation assays, we showed that nuclear corepressor and silencing mediator for retinoid and thyroid receptors were associated with the PLA2g2a gene in the presence of T3. In contrast with the established role of T3 to promote coactivator association with TRβ, our experiments demonstrate a novel inverse recruitment mechanism in which liganded TRβ recruits corepressors to inhibit PLA2g2a expression.

Keywords: Carnitine Palmitoyltransferase; Corepressor Transcription; Liver; Nuclear Receptors; Phospholipase A; Thyroid Hormone; Thyroid Hormone Receptor; Transcriptional Regulation.

FIGURE 1.

T₃ decreases sPLA₂ expression. A, rat hepatocytes were treated with 100 nm thyroid hormone (T₃) for 24 h. PLA2g2a mRNA levels were measured by real time PCR. B, rat hepatocytes were treated with T₃ for various times, and PLA2g2a RNA abundance was measured. C, PLA2g2a protein in rat hepatocytes was assessed by Western blotting after 24 h T₃ treatment. D, hepatocytes were exposed to T₃ or actinomycin D or both as in B. RNA was assessed at various time points. E, PLA2g2a mRNA levels in HepG2 cells were assessed following exposure to T₃ for 24 h. F, PLA2g2a protein levels in HepG2 cells were measured by ELISA. The data are expressed as the relative RNA or protein expression. All of the experiments were repeated four to six times. The data are expressed as the means of the fold induction by T₃ ± S.E. of mRNA abundance relative to untreated cells. **, p < 0.01; ***, p < 0.001. Con, control.

FIGURE 2.

T₃ inhibits the TNFα and IL-6 induction of PLA2g2a. A, rat hepatocytes were treated with 25 ng/ml TNFα or 100 nm T₃ or both for 24 h. mRNA abundance was assessed as described for Fig. 1. B, HepG2 cells were treated with 10 ng/ml IL-6 or 100 nm T₃ for 24 h. RNA abundance was determined. C, medium was collected from HepG2 cells treated with IL-6 or T₃, and the PLA2g2a levels were determined by ELISA. All of the experiments were repeated four to six times. The data are expressed as the means of the fold induction ± S.E. of mRNA abundance relative to untreated control cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 3.

T₃ inhibits PLA2g2a in vivo. Rats were made hypothyroid with an iodine-free diet and the addition of PTU. After 5 weeks, the animals were given 0.33 mg/kg T₃, and mRNA was harvested from the liver. A–E, the mRNA levels were measured for PLA2g2a (A), PLA2g1b (B), PLA2g3 (C), PLA2g5 (D), and SREBP-1c (E). F, pyruvate dehydrogenase kinase 4 (PDK4). G, PEPCK. H, CPT1a. I, the protein abundance of PLA2g2a was determined by Western blotting. The values are the averages of RNA from four rats. The data are expressed as the means of the fold induction by PTU + T₃ ± S.E. of mRNA abundance relative to PTU-treated rats. **, p < 0.01; ***, p < 0.001.

FIGURE 4.

Localization of a T₃-responsive element. A, HepG2 cells were transfected with 2 μg of −448/+58 PLA2g2a luciferase, 1 μg of SV40-TRβ or TRβ-LBD, and 0.1 μg of TK-Renilla and treated with or without T₃ for 24 h. B, HepG2 cells were transfected with serial deletions of the rat PLA2g2a promoter ligated in front of the luciferase reporter gene (PLA2g2a-luc) and an expression vector for TRβ. The cells were treated with T₃ for 24 h. The data are expressed as relative inhibition with T₃. All transfections were repeated four to six times. The significance is calculated relative to the empty vector PGL4. The error bars indicate S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001). The immobilized template assay was conducted with the biotinylated DNA corresponding to region −119/+58 and +1158/+1256 control region. C, schematic representation of PLA2g2a core promoter primers and control primers used to generate biotinylated DNA. The biotinylated DNA was incubated with histidine-tagged TRβ and histidine-tagged RXRα with and without T₃. The protein DNA complexes were resolved on Bis-Tris 4–12% gel and probed for His-TRβ using a TRβ antibody.

FIGURE 5.

Characterization of nTRE. A, double-stranded oligonucleotides were constructed that encompassed the −102/−82 nucleotides in the PLA2g2a gene. The ³²P-radiolabeled double-stranded oligomer representing the −102/−82 element was incubated with purified TRβ and RXRα. Antibodies to TRβ and IgG as well as 100 nm of T₃ were added. B, to assess the specificity of the TR-RXRα binding, a 10-fold excess of the competitor oligomers was added. Competition assays were conducted using double-stranded unlabeled −102/−82, direct repeat of AGGTCA separated by four nucleotides (DR4) and nonspecific oligomer (N.S). C, the −102/−82 element was cloned into a luciferase reporter plasmid in front of the SV40 promoter (−102/−82 SV40-luc). This reporter was cotransfected with TRβ into HepG2 cells in the presence or absence of T₃. The cells were treated with T₃ for 24 h. The transfections were repeated four times. Luciferase activity was corrected for both protein content and Renilla activity. The error bar indicates S.E. **, p < 0.01.

FIGURE 6.

Identification of nucleotides critical for T₃ responsiveness. Mutations in the −102/−82 element were made to identify the nucleotides necessary for TRβ binding. A, the sequence of the various nucleotide substitutions is shown. B, competition gel shift assays were conducted with a 10-fold excess of cold −102/−82 mutants and purified TRβ and RXRα proteins. C, different mutants of PLA2g2a promoter corresponding to the gel shift oligomer sequences were introduced by site-directed mutagenesis. HepG2 cells were transiently transfected with 2 μg of different PLA2g2a luciferase mutants, 1 μg of SV40-TRβ, and 0.1 μg of TK-Renilla. The cells were treated with or without T₃ for 24 h. D, HepG2 cells were transfected with PLA2g2a-luc and treated with 25 ng/ml TNFα or 100 nm T₃ as described for C. All transfections were repeated four times. Luciferase activity was corrected for both protein content and Renilla activity. The error bar indicates S.E. *, p < 0.05; **, p < 0.01.

FIGURE 7.

Coactivators participate in the induction of PLA2g2a by unliganded TRβ. A, HepG2 cells were transiently transfected with 2 μg of PLA2g2a luciferase reporters, 1 μg of null vector pSV-sport or SV40-TRβ, and 0.1 μg of TK-Renilla. T₃ was added for 24 h. B, HepG2 cells were transiently transfected with 2 μg of PLA2g2a luciferase reporters, 1 μg of VP16-TRβ and 0.1 μg of TK-Renilla. T₃ was added for 24 h. C, TREX2 SV40-luc was tested with VP16-TRβ as above. D, PLA2g2a-luc was cotransfected with expression vectors for TRβ or CBP. T₃ was added for 24 h. E, PLA2g2a was transfected with three different TRβ vectors carrying single amino acid substitutions. F, −4495/+1240 CPT1a-luc was transfected with the same TRβ expression vectors. The data are expressed as the relative luciferase activity. Luciferase activity was corrected for both protein content and Renilla activity. All of the experiments were repeated three to four times. The error bar indicates S.E. Significance is calculated relative wild type TRβ treated with T₃. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 8.

Corepressors are involved in T₃-mediated repression of PLA2g2a. HepG2 cells were transfected with siNCoR1 and siSMRT or scrambled siRNA overnight. The following day, the cells were treated with 100 nm T₃ for 24 h. A, the knockdown of NCoR1 mRNA was assessed by real time PCR. B, the knockdown of SMRT RNA abundance was measured. C and D, the protein abundance of NCoR1 (C) and SMRT (D) following knockdown is shown. E, the mRNA abundance of PLA2g2a was measured. F, the mRNA levels of the PEPCK gene were measured. G, the protein abundance of PLA2g2a in the media was measured by ELISA. The data are expressed as the relative expression ± S.E. of mRNA abundance of T₃ and untreated HepG2 cells. *, p < 0.01 to 0.05; **, p < 0.01; ***, p < 0.001. Scr, scrambled.

FIGURE 9.

Corepressors are recruited to the PLA2g2a gene by T₃. Hepatocytes were treated with T₃ for 24 h. The cells were cross-linked, and the DNA was sheared for chromatin immunoprecipitation assays. A, a model of the PLA2g2a promoter, and the locations of the primers are shown. B, the TRβ antibody was used for immunoprecipitation. PCR products for the proximal promoter and the third intron are shown. C, ChIP experiments were conducted with an antibody to NCoR1. D, ChIP experiments were carried out with a SMRT antibody. Con, control.

FIGURE 10.

Corepressors participate in the T₃ inhibition of several hepatic genes. HepG2 cells were treated with siNCoR1 or siSMRT as in Fig. 8. Cells were treated with 100 nm T₃ for 24 h. The mRNA abundance was determined by real time PCR. A, AKAP-4; B, SERPINE; C, S SLCA4; D, STK17-B; E, SLC26A3; F, HS3T3A1; G, FAM46A; H, SLC2A1; I, KIAA1199; J, CD24; K, SCGN; L, SORBS1. The data are expressed as the relative expression ± S.E. of mRNA abundance of T₃ and untreated HepG2 cells. *, p < 0.01 to 0.05; **, p < 0.01; ***, p < 0.001. Scr, scrambled.