Abstract
Retinoic acid (RA) and thyroid hormone are critical for differentiation and organogenesis in the embryo. Mct8 (monocarboxylate transporter 8), expressed predominantly in the brain and placenta, mediates thyroid hormone uptake from the circulation and is required for normal neural development. RA induces differentiation of F9 mouse teratocarcinoma cells toward neurons as well as extraembryonal endoderm. We hypothesized that Mct8 is functionally expressed in F9 cells and induced by RA. All-trans-RA (tRA) and other RA receptor (RAR) agonists dramatically (>300-fold) induced Mct8. tRA treatment significantly increased uptake of triiodothyronine and thyroxine (4.1- and 4.3-fold, respectively), which was abolished by a selective Mct8 inhibitor, bromosulfophthalein. Sequence inspection of the Mct8 promoter region and 5'-rapid amplification of cDNA ends PCR analysis in F9 cells identified 11 transcription start sites and a proximal Sp1 site but no TATA box. tRA significantly enhanced Mct8 promoter activity through a consensus RA-responsive element located 6.6 kilobases upstream of the coding region. A chromatin immunoprecipitation assay demonstrated binding of RAR and retinoid X receptor to the RA response element. The promotion of thyroid hormone uptake through the transcriptional up-regulation of Mct8 by RAR is likely to be important for extraembryonic endoderm development and neural differentiation. This finding demonstrates cross-talk between RA signaling and thyroid hormone signaling in early development at the level of the thyroid hormone transporter.
Highlights
Retinoic acid (RA) and thyroid hormones are essential for vertebrate development [1, 2]
Thyroid hormone is critical for brain development, and neural differentiation in F9 cells should be accompanied by induction of thyroid hormone transporter(s)
Induction of Mct8 mRNA by RA in F9 Cells—tRA treatment markedly increased Mct8 mRNA expression in F9 cells grown in media with 10% serum
Summary
Cell Culture—F9 cells, purchased from ATCC (Manassas, VA), were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) (Invitrogen) in gelatin-coated flasks or Petri dishes as recommended. Quantitative PCR of mouse thyroid hormone transporter genes, glyceraldehyde-3phosphate dehydrogenase gene (Gapdh), 18 S ribosomal RNA, and human MCT8 mRNA was performed with custom DNA primers synthesized by Invitrogen (supplemental Table 1). Grown in 12-well plates, as well as empty wells for measurement of nonspecific binding of radiolabeled thyroid hormone to the surface of the side wall of the well were rinsed with 1 ml of Dulbecco’s PBS, preincubated with 300 l of Hanks’ balanced salt solution with 0.1% bovine serum albumin (BSA) for 15 min at 37 °C, and the medium was replaced with 300 l of preheated thyroid hormone uptake assay buffer. Eluted DNA, as well as the aliquot of sheared chromatin prior to immunoprecipitation (input), was amplified by using the Expand high fidelity PCR system (Roche Applied Science) with primers specific to an RA-responsive region of mouse Mct or the mouse Lat (L-type amino acid transporter 1) promoter (supplemental Table 1). The amplicons were analyzed by electrophoresis in a 2% agarose gel
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