Sterol Regulatory Element-Binding Proteins Are Regulators of the NIS Gene in Thyroid Cells

Abstract

The uptake of iodide into the thyroid, an essential step in thyroid hormone synthesis, is an active process mediated by the sodium-iodide symporter (NIS). Despite its strong dependence on TSH, the master regulator of the thyroid, the NIS gene was also reported to be regulated by non-TSH signaling pathways. In the present study we provide evidence that the rat NIS gene is subject to regulation by sterol regulatory element-binding proteins (SREBPs), which were initially identified as master transcriptional regulators of lipid biosynthesis and uptake. Studies in FRTL-5 thyrocytes revealed that TSH stimulates expression and maturation of SREBPs and expression of classical SREBP target genes involved in lipid biosynthesis and uptake. Almost identical effects were observed when the cAMP agonist forskolin was used instead of TSH. In TSH receptor-deficient mice, in which TSH/cAMP-dependent gene regulation is blocked, the expression of SREBP isoforms in the thyroid was markedly reduced when compared with wild-type mice. Sterol-mediated inhibition of SREBP maturation and/or RNA interference-mediated knockdown of SREBPs reduced expression of NIS and NIS-specific iodide uptake in FRTL-5 cells. Conversely, overexpression of active SREBPs caused a strong activation of the 5'-flanking region of the rat NIS gene mediated by binding to a functional SREBP binding site located in the 5'-untranslated region of the rat NIS gene. These findings show that TSH acts as a regulator of SREBP expression and maturation in thyroid epithelial cells and that SREBPs are novel transcriptional regulators of NIS.

Sterol regulatory element-binding proteins are transcriptional regulators of the thyroglobulin gene in thyroid cells

“…HepG2 cells were cultured with RMPI 1640 medium supplemented with 10% FBS and FRTL-5 cells with Ham's F12 medium supplemented with 5% NCS and a six-hormone mixture ("6 hormone medium") as described in our recent publication [30]. Medium was changed every 2 days.…”

“…Following treatment, total RNA was extracted from cells using Trizol reagent (Life Technologies, Darmstadt, Germany) according to the manufacturer's protocol. cDNA synthesis and qPCR using gene-specific primers synthesized by Eurofins Genomics (Ebersberg, Germany) were performed as described recently in detail [30]. Features of genespecific primers are shown in Table 1.…”

“…Following treatment, cells were lysed with radioimmunoprecipitation assay lysis buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate] containing protease inhibitors (Sigma-Aldrich Chemie GmbH, Germany). Determination of protein content, protein separation by SDS-PAGE, electrotransfer to nitrocellulose membranes and blocking of membranes were carried out as described recently [30]. Afterwards, membranes were incubated overnight at 4°C with primary anti-TG antibody (rabbit polyclonal; 1:500 dilution; Abcam, Cambridge, UK) or anti-β-actin antibody (mouse monoclonal; 1:10.000 dilution; Abcam) as a reference protein for normalization.…”

“…The membranes were washed, and then incubated with a horseradish peroxidase-conjugated secondary anti-IgG antibody (rabbit polyclonal or mouse monoclonal; 1:10.000 dilution; Sigma-Aldrich and Abcam, respectively) at room temperature. Blot development and detection by chemiluminescence were performed as described recently [30]. For calculation of normalized protein concentrations, the band intensity of TG was normalized by that of β-actin.…”

“…Despite the outstanding role of the TSH-dependent signaling pathway in regulating genes involved in TH synthesis, it was reported that NIS, TPO and TG are also transcriptionally regulated by TSH-independent pathways like the NF-kB pathway or non-TSH signals like lipopolysaccharide [27][28][29]. In this context it was moreover shown that NIS and TPO are up-regulated by sterol regulatory element-binding proteins (SREBP)-1c and -2 via specific SREBP binding motifs, called sterol response elements (SRE), in their regulatory region [30,31]. SREBP were initially discovered as master transcription factors of cholesterol and fatty acid synthesis [32].…”