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

Abstract

Sterol regulatory element-binding proteins (SREBPs)-1c and -2, which were initially discovered as master transcriptional regulators of lipid biosynthesis and uptake, were recently identified as novel transcriptional regulators of the sodium-iodide symporter gene in the thyroid, which is essential for thyroid hormone synthesis. Based on this observation that SREBPs play a role for thyroid hormone synthesis, we hypothesized that another gene involved in thyroid hormone synthesis, the thyroid peroxidase (TPO) gene, is also a target of SREBP-1c and -2. Thyroid epithelial cells treated with 25-hydroxycholesterol, which is known to inhibit SREBP activation, had about 50% decreased mRNA levels of TPO. Similarly, the mRNA level of TPO was reduced by about 50% in response to siRNA mediated knockdown of both, SREBP-1 and SREBP-2. Reporter gene assays revealed that overexpression of active SREBP-1c and -2 causes a strong transcriptional activation of the rat TPO gene, which was localized to an approximately 80 bp region in the intron 1 of the rat TPO gene. In vitro- and in vivo-binding of both, SREBP-1c and SREBP-2, to this region in the rat TPO gene could be demonstrated using gel-shift assays and chromatin immunoprecipitation. Mutation analysis of the 80 bp region of rat TPO intron 1 revealed two isolated and two overlapping SREBP-binding elements from which one, the overlapping SRE+609/InvSRE+614, was shown to be functional in reporter gene assays. In connection with recent findings that the rat NIS gene is also a SREBP target gene in the thyroid, the present findings suggest that SREBPs may be possible novel targets for pharmacological modulation of thyroid hormone synthesis.

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

“…FRTL-5 cells with a targeted knockdown of SREBP-1c or SREBP-2 were generated by transfection of gene-specific Stealth RNAi molecules (from Invitrogen, Karlsruhe, Germany) using LipofectAMINE 2000 (Invitrogen) as described recently [30,31]. Following transfection, FRTL-5 cells were incubated with 6 hormone medium for 24 h and then total RNA extraction and cell lysis, respectively, was carried out as described above.…”

“…After reaching a confluence of 70-80%, cells were transiently transfected with the generated TG reporter gene constructs and cotransfected with either rat nSREBP-1c or nSREBP-2 expression plasmids or the empty expression plasmid (pcDNA3.1) as described recently [30,31]. The only modification was that both cell lines were co-transfected with 5 ng of pGL4.74 [hRluc/TK] (containing a renilla luciferase reporter gene and an HSV-TK promoter) as an internal control reporter vector to normalize for differences in transfection efficiency.…”

“…EMSA was performed with recombinant rat nSREBP-1c and nSREBP-2, in vitro-translated from the corresponding expression plasmids, as described recently [30,31]. Annealed oligonucleotides spanning either the wild-type or the mutated E-box SRE − 63 of rat TG promoter region were end-labeled with Digoxigenin (DIG) using the DIG Gel Shift Kit, 2nd Generation (Roche Diagnostics, Mannheim, Germany).…”

“…In addition, annealed and DIGlabeled oligonucleotides spanning either the wild-type or the mutated human LDLR-SRE were used as specific and non-specific control. Oligonucleotide sequences of specific and non-specific control have been described recently [30,31]. For competition experiments, recombinant nSREBP-1c and nSREBP-2, respectively, were incubated with DIG-labeled rat TG E-box SRE − 63 and increasing molar excess (5-, 50-and 100-fold) of unlabeled specific probe (human LDLR-SRE).…”

“…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].…”