PMC

Activity of the Mct8 proximal promoter in F9 cells. Cells were transfected with the indicated Luc reporter vector and pRL-CMV, treated with or without tRA (1 μm) for 24 h, and harvested for the luciferase assay. Data of pGL3 Basic in untreated F9 cells were set at 1. Values are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01, when compared with pGL3 Basic without tRA.

Effects of a transcription inhibitor, actinomycin D, on Mct8 mRNA expression in F9 cells. A, cells were treated with tRA (1 μm) and the indicated concentration of actinomycin D for 21 h, and quantitative RT-PCR of Mct8 was performed. Results were normalized to 18 S ribosomal RNA. Values are expressed as means ± S.D. (n = 3). *, p < 0.01, when compared with control (0 m). B, effects of tRA on degradation of Mct8 mRNA in F9 cells. To induce abundant Mct8, cells were pretreated with tRA (1 μm) for 24 h. Cells were rinsed twice with Dulbecco's PBS, cultured in growth media for 48 h, treated with actinomycin D (10 μm) for 1 h, and then treated with or without tRA (1 μm) in the presence of actinomycin D for the indicated time. Results of quantitative RT-PCR of Mct8 were normalized to 18 S ribosomal RNA. Data at 0 h were set at 100%. Values are expressed as means ± S.D. (n = 3).

Induction of Mct8 mRNA expression by retinoids in F9 cells. Results of quantitative RT-PCR for Mct8 and Gapdh mRNA are shown. A, time course of the induction of Mct8 mRNA by tRA. The inset graph shows induction within the first 24 h. Cells were treated with tRA (1 μm) for the indicated times. B, dose dependence of the induction of Mct8 mRNA by tRA. Cells were treated with the indicated concentration of tRA for 48 h. C, cells were treated with the indicated retinoid receptor agonist (1 μm) for 48 h (Re80 and Am80-RAR agonists, HX630 and PA024-RXR agonists). D, cells were treated with or without tRA (0.5 μm) and the indicated retinoid receptor antagonist (10 μm) for 48 h (LE135-RAR antagonist, HX531 and PA452-RXR antagonists). Treatment with tRA (1 μm) for 48 h was used to generate standard curves to quantify Mct8 and Gapdh. F9 cells were maintained in DMEM with 10% FBS. Values are expressed as means ± S.D. (error bars) (n = 3). *, p < 0.05; **, p < 0.01, when compared with untreated cells (A–C) or cells with only tRA (D).

Uptake of thyroid hormones by F9 cells. Cells were treated with or without tRA (1 μm) in the presence of 10% FBS for 6 days before the assay. A and B, time courses of T₃ accumulation (A) and T₄ accumulation (B). Cells were incubated with the ¹²⁵I-labeled T₃ uptake buffer (A) or ¹²⁵I-labeled T₄ uptake buffer (B) for the indicated times. Values are expressed as means ± S.D. (n = 4). *, p < 0.01, when compared with untreated cells. C, pharmacological inhibition of T₃ uptake. Cells were incubated with the ¹²⁵I-labeled T₃ uptake buffer for 10 min in the presence of BSP (Mct8 inhibitor), BCH (Lat inhibitor), or probenecid (Prob, pan-Oatp inhibitor) (1 mm each). Values are expressed as means ± S.D. (n = 3). *, p < 0.05, when compared with +tRA/−inhibitor. **, p < 0.02, when compared with −tRA/−inhibitor or +tRA/+BCH. D, dose dependence of BSP inhibition of tRA-induced T₃ uptake. Cells were incubated with ¹²⁵I-labeled T₃ uptake buffer in the presence of BSP at the indicated concentrations. T₃ uptake without BSP was simultaneously measured in tRA-treated and -untreated cells. Uptake, normalized to protein content, without BSP or tRA was subtracted from uptake at each concentration of BSP in tRA-treated cells, and uptake without BSP in tRA-treated cells was set at 100%. Values are expressed as means ± S.D. (error bars) (n = 3). The best fit line (R² = 0.93) is shown.

Expression of differentiation markers and Mct8 mRNA in F9 cells. A–D, induction of markers of endoderm differentiation in F9 cells. Cells were grown in DMEM supplemented with 10% FBS and treated with or without tRA (1 μm) and/or 8-bromo-cAMP (1 mm) for 96 h, and quantitative RT-PCR of the indicated genes was performed. Quantification of those genes as well as the internal control Gapdh was obtained from standard curves of serial dilution series of cDNA mixture of tRA-treated cells and tRA/cAMP-treated cells. The sample quantification was then normalized to Gapdh. E–H, induction of neural differentiation markers in F9 cells. Cells were treated with or without tRA (1 μm) and 8-bromo-cAMP (1 mm) in DMEM/F-12 (50:50) with 2% FBS for 2 days or 7 days, and quantitative RT-PCR of the indicated gene was performed. Quantification was obtained from standard curves of serial dilution series of cDNA at day 2 and normalized to Gapdh. Values are expressed as means ± S.D. (n = 3). *, p < 0.01, when compared with untreated cells (day 0). Col4a1, collagen type IV α-1; Afp, α-fetoprotein, Lama1, laminin α1; Thbd, thromobomodulin; Mct8, monocarboxylate transporter 8; Snca, synuclein α; Otx1, orthodenticle homeobox 1; Nes, nestin; NeuroD1, neurogenic differentiation 1.

Characterization of mouse Mct8 RARE in F9 cells. A, location and sequence of putative RAREs in the mouse Mct8 locus. B, functional analysis of putative RAREs in F9 cells. Three putative RAREs were fused to a heterologous SV40 promoter in Luc reporter vector constructs (upper panel) and transfected into F9 cells with pRL-CMV. Cells were treated with or without tRA (1 μm) for 24 h, and the luciferase assay was performed. Normalized data of the pGL promoter (−tRA) were set at 1. C, enhancer activity of the DR5A RARE with the Mct8 proximal promoter. Cells were transfected with the indicated Luc reporter constructs as well as pRL-CMV, treated with or without tRA (1 μm) for 24 h, and harvested for the luciferase assay. Normalized data of pGL3 Basic (−tRA) were set at 1. D, mutation analysis of the DR5A RARE. In the left panel, mutated bases in the DR5A RARE are indicated by an asterisk. Cells were transfected with the mutated vector (pGL3 DR5A-M −836/−54) or the original vector with wild DR5A (pGL3 DR5A −836/−54) and treated with or without tRA (1 μm) for 24 h. Normalized data of pGL3 DR5A −836/−54 (−tRA) were set at 1. Values are means ± S.D. (n = 3). *, p < 0.01, when compared with −tRA in each vector. E, binding of endogenous retinoid receptors to the region of DR5A RARE. F9 cells were treated with tRA (1 μm) for the indicated time, and a ChIP assay was performed with the indicated antibodies or preimmune IgG. The DR5A region, as well as the tRA-unresponsive Lat1 promoter, was amplified by PCR from the immunoprecipitated (IP) chromatin and analyzed by agarose gel electrophoresis. Expected amplicon sizes were 217 and 203 bp, respectively. The DNA size marker indicates 300 and 200 bp.

TSS of Mct8 in F9 cells. A, results of 5′-RACE from tRA-treated F9 cells. Shown is agarose gel electrophoresis of the second round PCR with GeneRacer 5′ nested primer and 3′ +496 primer. Marker (M) was a 100-bp ladder. B, distribution of the TSS of Mct8 in F9 cells. The sequence of the 5′-flanking region of Mct8 is shown. The positions of the identified TSSs are marked with vertical lines, the height of which indicates the frequency of RACE clones found to initiate at each site. Putative core promoter elements are shown. DPE, downstream core promoter element. C, diagram of the promoter region and amplification of upstream transcripts, including the Sp1 site (−128) and downstream transcripts without the Sp1 site (−79). D, determination of the influence of tRA treatment on transcripts. RT-PCR of the 5′ region of Mct8 in F9 cells treated with or without tRA (1 μm) for 48 h is shown. The internal control, Gapdh, is shown on the right. E, comparison of TSS positions in the mouse and human MCT8 genes. Alternative first exons, CpG islands, canonical Sp1 sites (arrow), and homologous regions between the two species are shown. TSSs of mouse Mct8 as well as that of the short variation of human MCT8 flanked by AP2 (alternative promoter 2) (DBTSS) are located in CpG islands in high homologous regions (79%) between the two species. The long variation of human MCT8, flanked by AP1 (DBTSS), is transcribed from a human-specific region outside of the CpG island.