CD40, a tumor necrosis factor receptor, is a major immune-modulating susceptibility gene for Graves disease (GD) as well as for a variety of other autoimmune diseases. Its broad association with autoimmunity underscores its paramount role in the development of a normal adaptive immune response, primarily in coordinating effective antigen presentation. The molecular pathways by which CD40 activation in the thyroid induces GD are unknown. In this study, we investigated whether NF-κB, a ubiquitious family of transcription factors, mediates the downstream effects of thyroid-specific CD40 activation. Cultured primary human thyrocytes, from patients with and without GD, underwent CD40 stimulation. Once stimulated, cytokines and transcription factors specific for either the canonical nuclear factor κB (NF-κB)1 pathway [interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α], which primarily recruits cells for innate immunity, or the noncanonical NF-κB2 pathway [B cell-activating factor of the TNF family, CC chemokine ligand (CCL)21], which directs B cell viability, were analyzed. Significant upregulation in the messenger RNA and protein levels of both canonical and noncanonical pathway cytokines was observed. Western blot analyses of the specific transcription factors for the NF-κB1 and NF-κB2 pathways (p65 and p100/p52, respectively) demonstrated that p65 is constitutively expressed. In contrast, CD40 stimulation robustly increased the expression of the NF-κB2 p52 transcription factor, and the upregulation was significantly more profound in the GD tissue than in the normal thyroid tissue. Our data show that CD40 activity in thyrocytes is prominently mediated via NF-κB and furthermore suggest that the NF-κB1 and NF-κB2 pathways both contribute to the triggering and the progression of GD.

Thyroid hormone receptor β (TRβ) suppresses tumor growth through regulation of gene expression, yet the associated TRβ-mediated changes in chromatin assembly are not known. The chromatin ATPase brahma-related gene 1 (BRG1; SMARCA4), a key component of chromatin-remodeling complexes, is altered in many cancers, but its role in thyroid tumorigenesis and TRβ-mediated gene expression is unknown. We previously identified the oncogene runt-related transcription factor 2 (RUNX2) as a repressive target of TRβ. Here, we report differential expression of BRG1 in nonmalignant and malignant thyroid cells concordant with TRβ. BRG1 and TRβ have similar nuclear distribution patterns and significant colocalization. BRG1 interacts with TRβ, and together, they are part of the regulatory complex at the RUNX2 promoter. Loss of BRG1 increases RUNX2 levels, whereas reintroduction of TRβ and BRG1 synergistically decreases RUNX2 expression. RUNX2 promoter accessibility corresponded to RUNX2 expression levels. Inhibition of BRG1 activity increased accessibility of the RUNX2 promoter and corresponding expression. Our results reveal a mechanism of TRβ repression of oncogenic gene expression: TRβ recruitment of BRG1 induces chromatin compaction and diminishes RUNX2 expression. Therefore, BRG1-mediated chromatin remodeling may be obligatory for TRβ transcriptional repression and tumor suppressor function in thyroid tumorigenesis.

Instructive signals that delineate the formation of thyroid follicles by thyrotropin (TSH) in stem cells are complex. Here, we have examined the role of protein kinase C (PKC) by using a unique Gαq/11 biased small molecule (MSq1) to develop thyroid progenitor cells. Mouse embryonic stem cells (mESCs) were differentiated into anterior endoderm cells and treated with either TSH or MSq1 in the presence or absence of PKC inhibitors. The transcriptional and translational response of key thyroid markers-sodium iodide symporter (NIS), thyroglobulin (TG), and thyrotropin receptor (TSHR) as well as potential signaling molecules-were then analyzed. The data confirmed that MSq1 is a potent Gαq/11 activator with a major increase in Gαq/11 signaling when compared to TSH. MSq1 activation resulted in an increase in thyroid-specific genes, demonstrating that enhanced PKC signaling was able to induce their expression. The specificity of the PKC signals over the protein kinase A (PKA) pathway in regulating thyroid gene expression was shown by using a specific PKC enzyme inhibitor. The data revealed that TG and NIS expression were suppressed in the presence of the PKC inhibition but, in contrast, were not influenced by PKA inhibition. This indicated that PKC activation was the dominant pathway in the inductive process for thyroid hormone production. Furthermore, by examining PKC isoforms we found that PKCξ was the predominant form in the ES cells that mediated the effects. Since PKCξ can lead to activation of transforming growth factor-β-activated kinase (pTAK1), and its downstream effector nuclear factor κB (NFκB) complex, this demonstrated the involvement of the TAK1/NFκB pathway in thyroid speciation.

Papillary thyroid carcinoma (PTC) is the most prevalent endocrine-related malignancy. In spite of the good prognosis, a more aggressive disease can develop in some PTC patients, leading to poor survival. Nuclear paraspeckle assembly transcript 1 (NEAT1) enhances tumorigenesis; however, the relationship between NEAT1_2 and glycolysis in PTC has not been identified. The expressions of NEAT1_2, KDM5B, Ras-related associated with diabetes (RRAD), and EHF were determined by quantitative reverse transcription polymerase chain reaction and immunocytochemistry. The effects of NEAT1_2, KDM5B, RRAD, and EHF on PTC glycolysis were ascertained employing in vitro as well as in vivo experiments. Chromatin immunoprecipitation (ChIP), RNA binding protein immunoprecipitation, luciferase reporter assays, and co-immunoprecipitation were utilized to analyze the binding abilities among NEAT1_2, KDM5B, RRAD, and EHF. Overexpression of NEAT1_2 was associated with glycolysis in PTC. NEAT1_2 could activate glycolysis by regulating the expression of RRAD in PTC. NEAT1_2 mediated H3K4me3 modification at the promoter of RRAD by recruiting KDM5B. RRAD further negatively regulated glycolysis by binding and regulating the subcellular location of the transcription factor EHF. EHF could activate the transcription of NEAT1_2, hexokinase 2, and pyruvate kinase M2, thereby forming the NEAT1_2/RRAD/EHF feedback loop. Our study revealed that the NEAT1_2/RRAD/EHF positive feedback loop facilitated glycolysis in PTC, which might avail meaningful insight for PTC management.

Cellular retinoic acid-binding protein 2 (CRABP2) participates in retinoid partitioning between different nuclear receptors. Recently, we identified that CRABP2 is one of the progression-associated genes in thyroid cancer. To explore the prognostic and functional significance of CRABP2, immunohistochemical analysis was performed in thyroid tissues and neoplasms. Overexpression of CRABP2 was observed in malignant thyroid neoplasms but not in benign thyroid lesions. CRABP2 expression was an independent predictive factor for recurrence-free survival in patients with differentiated thyroid cancer. Knockdown of CRABP2 reduced the sensitivity of thyroid cancer cells to retinoic acid. Importantly, CRABP2 expression in thyroid cancer cells was associated with epithelial-mesenchymal transition properties, including anoikis resistance, migration, and invasion capacity. Furthermore, invasion promoted by CRABP2 was mediated at least partly by the integrin/focal adhesion kinase/AKT pathway. In summary, CRABP2 expression is upregulated in thyroid cancer with adverse prognostic implications. The invasion-stimulating effects appear independent of canonical retinoic acid signaling and may serve as a potential therapeutic target.

Increasing brown adipose tissue (BAT) activity is regarded as a potential treatment of obese, hyperglycemic patients with metabolic syndrome. Triiodothyronine (T3) is known to stimulate BAT activity by increasing mitochondrial uncoupling protein 1 (Ucp1) gene transcription, leading to increased thermogenesis and decreased body weight. Here we report our studies on the effects of T3 and glucose in two mouse models and in mouse immortalized brown preadipocytes in culture. We identified carbohydrate response element binding protein (ChREBP) as a T3 target gene in BAT by RNA sequencing and studied its effects in brown adipocytes. We found that ChREBP was upregulated by T3 in BAT in both hyperglycemic mouse models. In brown preadipocytes, T3 and glucose synergistically and dose dependently upregulated Ucp1 messenger RNA 1000-fold compared with low glucose concentrations. Additionally, we observed increased ChREBP and Ucp1 protein 11.7- and 19.9-fold, respectively, along with concomitant induction of a hypermetabolic state. Moreover, downregulation of ChREBP inhibited T3 and glucose upregulation of Ucp1 100-fold, whereas overexpression of ChREBP upregulated Ucp1 5.2-fold. We conclude that T3 and glucose signaling pathways coordinately regulate the metabolic state of BAT and suggest that ChREBP is a target for therapeutic regulation of BAT activity.

Hypothyroidism is associated with an increased occurrence of atherosclerosis, suggesting some protective role for thyroid hormones (THs). Hypercholesterolemia is one of the major risk factor to develop this disease. Here, we show that the well-known TH cholesterol lowering effect was dependent on TH nuclear receptor (TR)β liver activity. But most importantly, TRα was also shown to contribute of slowing down atherosclerosis progression via an independent mechanism. Introduction of TRα(0/0) deletion in the ApoE(-/-) background accelerated the appearance of plaques. Earlier cholesterol accumulation was detected in aorta macrophages, likely due to impaired cholesterol efflux. The IL-1β inflammatory cytokine was elevated in serum and macrophages in correlation with an activation of the AKT/nuclear factor κB pathway in these cells. Inhibition of AKT prevented inflammation and restored normal cholesterol efflux. Similar low-grade inflammation was identified in TRα(0/0) male mice. Thus, the mere absence of TRα is associated with elevated levels of cytokines likely responsible for cholesterol accumulation and atherosclerosis. This TRα protective activity should be relevant for other inflammatory pathologies.

Thyroid-stimulating hormone (TSH) treatment activates inhibitor of NF-κB/nuclear factor κB (IκB/NFκB) and extracellular signal-regulated kinase (ERK)-P38 in macrophages, but how these pathways are activated, and how they contribute to the proinflammatory effect of TSH on macrophages remain unknown. The TSH receptor (TSHR) is coupled to 4 subfamilies of G proteins (Gs, Gi/o, Gq/11, and G12/13) for its downstream signaling. This study investigated the G protein subtypes responsible for the proinflammatory effect of TSH on macrophages. qPCR showed that Gi2, Gi3, Gas, Gq, G11, G12, G13, and G15 are abundantly expressed by macrophages. The contribution of different G protein pathways to the proinflammatory effect was studied by the corresponding inhibitors or siRNA interference. While TSH-induced IκB phosphorylation was not inhibited by Gs inhibitor NF449, Gi inhibitor pertussis toxin, or Gq or G11 siRNA, it was blocked by phospholipase C inhibitor U73122 or G15 siRNA interference. TSH-induced ERK and P38 phosphorylation was blocked by G13 but not G12 siRNA interference. Interference of either G13 or G15 could block the proinflammatory effect of TSH on macrophages. The present study demonstrate that TSH activates macrophage inflammation by the G13/ERK-P38/Rho GTPase and G15/phospholipase C (PLC)/protein kinases C (PKCs)/IκB pathways.