1H). to DNA, protein, and/or lipid oxidative damage. Thus, it was hypothesized that Nrf2 should also possess important functions in keeping thyroid homeostasis. Ubiquitous and thyroid-specific male C57BL6J Nrf2 knockout (Nrf2-KO) mice were analyzed. Plasma and thyroids were harvested for evaluation of thyroid function tests by radioimmunoassays and of gene and protein manifestation by real-time polymerase chain reaction and immunoblotting, respectively. Nrf2-KO and Keap1-KO clones of the PCCL3 rat thyroid follicular cell collection were Lumefantrine generated using CRISPR/Cas9 technology and were utilized for gene and protein manifestation studies. Software-predicted Nrf2 binding sites within the thyroglobulin enhancer were validated by site-directed mutagenesis and chromatin immunoprecipitation. The study demonstrates Nrf2 mediates antioxidant transcriptional reactions in thyroid cells and protects the thyroid from oxidation induced by iodide overload. Remarkably, it was also found that Nrf2 has a dramatic impact on both the basal large quantity and the thyrotropin-inducible intrathyroidal large quantity of thyroglobulin (Tg), the precursor protein of thyroid hormones. This effect is definitely mediated by cell-autonomous rules of gene manifestation by Nrf2 via its direct binding to two evolutionarily conserved antioxidant response elements in an upstream enhancer. Yet, despite upregulating Tg levels, Nrf2 limits Tg iodination both under basal conditions and in response to extra iodide. Nrf2 exerts pleiotropic functions in the thyroid gland to couple cell stress defense mechanisms to iodide rate of metabolism and the thyroid hormone synthesis machinery, both under basal conditions and in response to extra iodide. gene manifestation by Nrf2 via two evolutionarily conserved AREs. Thus, Nrf2 couples cell stress defense mechanisms to iodide rate of metabolism and the thyroid hormone synthesis machinery. Methods Nrf2 knockout mice C57BL/6J Nrf2+/? mice (15) were from RIKEN BRC (Tsukuba, Japan). Nrf2 wild-type (WT) and knockout (Nrf2-KO) mice were generated by mating Nrf2+/? males and females. Offspring were genotyped, as previously explained (15). For iodide challenge, male WT and Nrf2-KO mice (three to four months aged) fed a standard diet were supplied with normal tap water with or without 0.05% sodium iodide (NaI) for seven days. Mice were housed in the animal facility of the University or college of Patras Medical School in heat-, light-, and humidity-controlled rooms having a 12-hour Lumefantrine light/dark cycle. All animal methods were approved by the local Institutional Review Table and were in accordance with European Percentage Directive 86/609/EEC. Nrf2 thyroid-specific KO mice Mice expressing Cre recombinase under control of the Pax8 locus (Pax8[Cre/+]) (23) MMP19 were crossed with Nrf2 flox/flox mice that harbor flox sites flanking the DNA-binding website (exon 5) of the gene (24). The producing Pax8(Cre/+)-Nrf2 flox/+ mice were backcrossed with Nrf2 flox/flox mice to obtain Pax8(Cre/+)-Nrf2 flox/flox mice, hereafter referred to as thyroid-specific Nrf2 KO (ts-KO). and alleles were genotyped by polymerase chain reaction (PCR) using primers and conditions explained Lumefantrine in Supplementary Furniture S1CS4 (Supplementary Data are available on-line at www.liebertpub.com/thy). Thyroid-specific disruption as a result of recombination of the Nrf2 floxed allele was confirmed by genotyping thyroid DNA. Lumefantrine Real-time reverse transcription (RT)-PCR was also used to confirm the thyroid-specific deletion using primers focusing on the exon 5 of (Supplementary Table S5). Nrf2 flox/flox mice were used like a control group in experiments. Mice were housed.