A Simple Protocol for High Efficiency Protein Isolation After RNA Isolation from Mouse Thyroid and Other Very Small Tissue Samples.

As a dedicated hormone-secreting organ, the thyroid gland possesses a complement of proteostatic systems, including antioxidant, unfolded protein, and autophagic responses. The vast majority of animal investigations of thyroid physiology and, more recently, proteostasis, have utilized as model the rat, rather than the mouse. This is due to the very small size of the thyroid gland in the latter, with a total weight of ~2 mg (~1 mg per thyroid lobe). However, this strategy has limited the utilization of genetic approaches, such as taking advantage of the various transgenic and knockout mouse models. Here, we describe a simple and highly efficient protocol for the simultaneous isolation of mRNA, micro-RNA and 150-200 μg of protein from as little as 1 mg of mouse thyroid tissue, the average weight of one of the two thyroid lobes, thus preserving the other lobe for immunohistochemical or other analyses. While our workflow is similar to other protocols published in the literature and/or proposed by commercial reagent providers, we have introduced a key modification that addresses efficiently the most challenging step of the protein isolation process: the solubilization of the protein pellet after RNA extraction and protein precipitation. We demonstrate the feasibility of our approach and its utility for downstream analyses (including Western blotting) that facilitate the comparative study of proteostatic pathways in the mouse thyroid. We have also successfully applied this protocol on samples from mouse liver, brown and white adipose tissue, as well as from rodent cell lines

Emerging roles of Keap1/Nrf2 signaling in the thyroid gland and perspectives for bench-to-bedside translation.

The signaling pathway centered on the transcription factor nuclear erythroid factor 2-like 2 (Nrf2) has emerged during the last 15 years as a target for the prevention and treatment of diseases broadly related with oxidative stress such as cancer, neurodegenerative and metabolic diseases. The roles of Nrf2 are expanding beyond general cytoprotection, and they encompass its crosstalk with other pathways as well as tissue-specific functions. The thyroid gland relies on reactive oxygen species for its main physiological function, the synthesis and secretion of thyroid hormones. A few years ago, Nrf2 was characterized as a central regulator of the antioxidant response in the thyroid, as well as of the transcription and processing of thyroglobulin, the major thyroidal protein that serves as the substrate for thyroid hormone synthesis. Herein, we summarize the current knowledge about the roles of Nrf2 in thyroid physiology, pathophysiology and disease. We focus specifically on the most recent publications in the field, and we discuss the implications for the preclinical and clinical use of Nrf2 modulators

Patent Review (2017-2020) of the Keap1/Nrf2 Pathway Using PatSeer Pro: Focus on Autoimmune Diseases.

Research on the antioxidant pathway comprising the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and its cytoplasmic inhibitor Kelch-like ECH-associated protein 1 (Keap1) is ever increasing. As modulators of this pathway have started to be used in clinical trials and clinical practice, Nrf2 has become the subject of several patents. To assess the patent landscape of the last three years on Nrf2 and evaluate the main fields they refer to, we used the web-based tool PatSeer Pro to identify patents mentioning the Nrf2 pathway between January 2017 and May 2020. This search resulted in 509 unique patents that focus on topics such as autoimmune, neurodegenerative, liver, kidney, and lung diseases and refer to modulators (mainly activators) of the Nrf2 pathway as potential treatments. Autoimmunity emerged as the main theme among the topics of Nrf2 patents, including a broad range of diseases, such as systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel diseases, Hashimoto's thyroiditis, etc.; however, there was a dearth of experimental support for the respective patents' claims. Given that chronic inflammation is the main element of the pathophysiology of most autoimmune diseases, the majority of patents referring to activation of Nrf2 as a method to treat autoimmune diseases base their claims on the well-established anti-inflammatory role of Nrf2. In conclusion, there is strong interest in securing intellectual property rights relating to the potential use of Nrf2 pathway activators in a variety of diseases, and this trend parallels the rise in related research publications. However, in the case of autoimmunity, more research is warranted to support the potential beneficial effects of Nrf2 modulation in each disease

The Keap1/Nrf2 Signaling Pathway in the Thyroid-2020 Update.

The thyroid gland has a special relationship with oxidative stress. On the one hand, like all other tissues, it must defend itself against reactive oxygen species (ROS). On the other hand, unlike most other tissues, it must also produce reactive oxygen species in order to synthesize its hormones that contribute to the homeostasis of other tissues. The thyroid must therefore also rely on antioxidant defense systems to maintain its own homeostasis in the face of continuous self-exposure to ROS. One of the main endogenous antioxidant systems is the pathway centered on the transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2) and its cytoplasmic inhibitor Kelch-like ECH-associated protein 1 (Keap1). Over the last few years, multiple links have emerged between the Keap1/Nrf2 pathway and thyroid physiology, as well as various thyroid pathologies, including autoimmunity, goiter, hypothyroidism, hyperthyroidism, and cancer. In the present mini-review, we summarize recent studies shedding new light into the roles of Keap1/Nrf2 signaling in the thyroid

Transcriptomic profiling of the response to excess iodide in Keap1 hypomorphic mice reveals new gene-environment interactions in thyroid homeostasis.

Iodide plays a pivotal role in thyroid homeostasis due to its crucial involvement in thyroid hormone biosynthesis. Exposure to pharmacological doses of iodide elicits in the thyroid an autoregulatory response to preserve thyroid function, as well as an antioxidant response that is mediated by the Keap1/Nrf2 signaling pathway. The objective of the present study was to investigate the transcriptional response of the thyroid to excess iodide in a background of enhanced Nrf2 signaling. Keap1 knockdown (Keap1 <sup>KD</sup> ) mice that have activated Nrf2 signaling were exposed or not to excess iodide in their drinking water for seven days and compared to respective wild-type mice. RNA-sequencing of individual mouse thyroids identified distinct transcriptomic patterns in response to iodide, with Keap1 <sup>KD</sup> mice showing an attenuated inflammatory response, altered thyroidal autoregulation, and enhanced cell growth/proliferative signaling, as confirmed also by Western blotting for key proteins involved in antioxidant, autoregulatory and proliferative responses. These findings underscore novel gene-environment interactions between the activation status of the Keap1/Nrf2 antioxidant response system and the dietary iodide intake, which may have implications not only for the goiter phenotype of Keap1 <sup>KD</sup> mice but also for humans harboring genetic variations in KEAP1 or NFE2L2 or treated with Nrf2-modulating drugs

Broccoli sprout beverage is safe for thyroid hormonal and autoimmune status: Results of a 12-week randomized trial.

Sulforaphane is a redox-active natural product present in cruciferous vegetables like broccoli. Broccoli sprout-derived products are promising agents for the prevention of oxidative stress-related diseases, but some have long been suspected of thyroidal toxicity. Recent findings also raise the possibility that long-term exposure to sulforaphane, or to other natural substances or drugs that modulate the activity of the transcription factor Nrf2 (NFE2-related factor 2) may lead to thyroid dysfunction or thyroid autoimmune disease, questioning the safety of trials with sulforaphane-containing products. Previous studies addressing possible effects of sulforaphane-related compounds from natural product extracts on the thyroid were quite short and/or inconsistent. To investigate whether long-term exposure to a beverage enriched with sulforaphane and its precursor glucoraphanin may affect thyroid function, we analyzed biochemical measures of thyroid function and thyroid autoimmunity in 45 female participants in a randomized clinical trial at baseline and after 84 days of beverage administration. Serum levels of thyroid-stimulating hormone, free thyroxine and thyroglobulin were not affected by the treatment, and neither was the thyroid autoimmunity status of participants. These results provide evidence in favor of the safety of chemoprevention strategies that target the activation of Nrf2 to protect against environmental exposures and other oxidative stress-related pathologies

A Bibliometric Review of the Keap1/Nrf2 Pathway and its Related Antioxidant Compounds.

Nrf2 is a master transcriptional regulator of antioxidant and cytoprotective pathways. Currently in its third decade, research on Nrf2 has expanded to encompass not only basic but also clinical studies. In the present bibliometric review, we employed the VOSviewer tool to describe the existing Nrf2 literature landscape. As of July 2019, 11,931 papers on Nrf2 were listed in the "Web of Science" database, with more than 1000 new papers published each year. As expected, terms related to oxidative stress and antioxidant molecules occur very often in the Nrf2 literature throughout the years. Interestingly, there is also a gradual increase in the occurrence of terms related to diseases or to natural compounds, the most prominent being sulforaphane, curcumin, and resveratrol that modulate the Nrf2 pathway. Going beyond molecular biology/biochemistry and related fields, Nrf2 research has begun to spread into more clinical areas like endocrinology/metabolism, cardiology, and nephrology, likely reflecting an increased interest in clinical applications of Nrf2 pathway activators. China has become the most prolific producer of Nrf2 papers the last five years followed by the USA and Japan, a reverse pattern compared to the past. In conclusion, Nrf2 is the subject of a globally active research field that keeps growing and extends from bench to bedside

Dexamethasone Administration in Mice Leads to Less Body Weight Gain over Time, Lower Serum Glucose, and Higher Insulin Levels Independently of NRF2.

Glucocorticoids are used widely on a long-term basis in autoimmune and inflammatory diseases. Their adverse effects include the development of hyperglycemia and osteoporosis, whose molecular mechanisms have been only partially studied in preclinical models. Both these glucocorticoid-induced pathologies have been shown to be mediated at least in part by oxidative stress. The transcription factor nuclear erythroid factor 2-like 2 (NRF2) is a central regulator of antioxidant and cytoprotective responses. Thus, we hypothesized that NRF2 may play a role in glucocorticoid-induced metabolic disease and osteoporosis. To this end, WT and Nrf2 knockout (Nrf2KO) mice of both genders were treated with 2 mg/kg dexamethasone or vehicle 3 times per week for 13 weeks. Dexamethasone treatment led to less weight gain during the treatment period without affecting food consumption, as well as to lower glucose levels and high insulin levels compared to vehicle-treated mice. Dexamethasone also reduced cortical bone volume and density. All these effects of dexamethasone were similar between male and female mice, as well as between WT and Nrf2KO mice. Hepatic NRF2 signaling and gluconeogenic gene expression were not affected by dexamethasone. A 2-day dexamethasone treatment was also sufficient to increase insulin levels without affecting body weight and glucose levels. Hence, dexamethasone induces hyperinsulinemia, which potentially leads to decreased glucose levels, as well as osteoporosis, both independently of NRF2

Keap1/Nrf2 Signaling: A New Player in Thyroid Pathophysiology and Thyroid Cancer.

The Keap1/Nrf2 pathway is a key mediator of general redox and tissue-specific homeostasis. It also exerts a dual role in cancer, by preventing cell transformation of normal cells but promoting aggressiveness, and drug resistance of malignant ones. Although Nrf2 is well-studied in other tissues, its roles in the thyroid gland are only recently emerging. This review focuses on the involvement of Keap1/Nrf2 signaling in thyroid physiology, and pathophysiology in general, and particularly in thyroid cancer. Studies in mice and cultured follicular cells have shown that, under physiological conditions, Nrf2 coordinates antioxidant defenses, directly increases thyroglobulin production and inhibits its iodination. Increased Nrf2 pathway activation has been reported in two independent families with multinodular goiters due to germline loss-of-function mutations in KEAP1. Nrf2 pathway activation has also been documented in papillary thyroid carcinoma (PTC), due to somatic mutations, or epigenetic modifications in KEAP1, or other pathway components. In PTC, such Nrf2-activating KEAP1 mutations have been associated with tumor aggressiveness. Furthermore, polymorphisms in the prototypical Nrf2 target genes NQO1 and NQO2 have been associated with extra-thyroidal extension and metastasis. More recently, mutations in the Nrf2 pathway have also been found in Hürthle-cell (oncocytic) thyroid carcinoma. Finally, in in vitro, and in vivo models of poorly-differentiated, and undifferentiated (anaplastic) thyroid carcinoma, Nrf2 activation has been associated with resistance to experimental molecularly-targeted therapy. Thus, Keap1/Nrf2 signaling is involved in both benign and malignant thyroid conditions, where it might serve as a prognostic marker or therapeutic target