NR4A ablation improves mitochondrial fitness for long persistence in human CAR-T cells against solid tumors.

BACKGROUND: Antitumor effect of chimeric antigen receptor (CAR)-T cells against solid tumors is limited due to various factors, such as low infiltration rate, poor expansion capacity, and exhaustion of T cells within the tumor. NR4A transcription factors have been shown to play important roles in T-cell exhaustion in mice. However, the precise contribution of each NR4a factor to human T-cell differentiation remains to be clarified. METHODS: In this study, we deleted NR4A family factors, NR4A1, NR4A2, and NR4A3, in human CAR-T cells recognizing human epidermal growth factor receptor type 2 (HER2) by using the CRISPR/Cas9 system. We induced T-cell exhaustion in these cells in vitro through repeated co-culturing of CAR-T cells with Her2+A549 lung adenocarcinoma cells and evaluated cell surface markers such as memory and exhaustion phenotypes, proliferative capacity, cytokine production and metabolic activity. We validated the antitumor toxicity of NR4A1/2/3 triple knockout (TKO) CAR-T cells in vivo by transferring CAR-T cells into A549 tumor-bearing immunodeficient mice. RESULTS: Human NR4A-TKO CAR-T cells were resistant against exhaustion induced by repeated antigen stimulation in vitro, and maintained higher tumor-killing activity both in vitro and in vivo compared with control CAR-T cells. A comparison of the effectiveness of NR4A single, double, and TKOs demonstrated that triple KO was the most effective in avoiding exhaustion. Furthermore, a strong enhancement of antitumor effects by NR4A TKO was also observed in T cells from various donors including aged persons. Mechanistically, NR4A TKO CAR-T cells showed enhanced mitochondrial oxidative phosphorylation, therefore could persist for longer periods within the tumors. CONCLUSIONS: NR4A factors regulate CAR-T cell persistence and stemness through mitochondrial gene expression, therefore NR4A is a highly promising target for the generation of superior CAR-T cells against solid tumors.

NR4A3 prevents diabetes induced atrial cardiomyopathy by maintaining mitochondrial energy metabolism and reducing oxidative stress.

BACKGROUND: Atrial cardiomyopathy (ACM) is responsible for atrial fibrillation (AF) and thromboembolic events. Diabetes mellitus (DM) is an important risk factor for ACM. However, the potential mechanism between ACM and DM remains elusive. METHODS: Atrial tissue samples were obtained from patients diagnosed with AF or sinus rhythm (SR) to assess alterations in NR4A3 expression, and then two distinct animal models were generated by subjecting Nr4a3-/- mice and WT mice to a high-fat diet (HFD) and Streptozotocin (STZ), while db/db mice were administered AAV9-Nr4a3 or AAV9-ctrl. Subsequently, in vivo and in vitro experiments were conducted to assess the impact of NR4A3 on diabetes-induced atrial remodeling through electrophysiological, biological, and histological analyses. RNA sequencing (RNA-seq) and metabolomics analysis were employed to unravel the downstream mechanisms. FINDINGS: The expression of NR4A3 was significantly decreased in atrial tissues of both AF patients and diabetic mice compared to their respective control groups. NR4A3 deficiency exacerbated atrial hypertrophy and atrial fibrosis, and increased susceptibility to pacing-induced AF. Conversely, overexpression of NR4A3 alleviated atrial structural remodeling and reduced AF induction rate. Mechanistically, we confirmed that NR4A3 improves mitochondrial energy metabolism and reduces oxidative stress injury by preserving the transcriptional expression of Sdha, thereby exerting a protective influence on atrial remodeling induced by diabetes. INTERPRETATION: Our data confirm that NR4A3 plays a protective role in atrial remodeling caused by diabetes, so it may be a new target for treating ACM. FUNDING: This study was supported by the major research program of National Natural Science Foundation of China (NSFC) No: 82370316 (to Q-S. W.), No. 81974041 (to Y-P. W.), and No. 82270447 (to Y-P. W.) and Fundation of Shanghai Hospital Development Center (No. SHDC2022CRD044 to Q-S. W.).

Thyroid Hormone Receptors in Control of Heart Rate.

Thyroid hormone has profound effects on cardiovascular functions, including heart rate. These effects can be mediated directly, for example, by changing the expression of target genes in the heart through nuclear thyroid hormone receptors, or indirectly by altering the autonomic nervous systems output of the brain. The underlying molecular mechanisms as well as the cellular substrates, however, are far from being understood. In this review, we summarize the recent key findings on the individual contributions of the two thyroid hormone receptor isoforms on the regulation of heart rate, challenging the role of the pacemaker channel genes Hcn2 and Hcn4 as sole mediators of the hormone's effect. Furthermore, we discuss the possible actions of thyroid hormone on the autonomic nervous system affecting heart rate distribution, and highlight the possibility of permanent alterations in heart and brain by impaired thyroid hormone action during development as important factors to consider when analyzing or designing experiments.

A pan-cancer analysis unveiling the function of NR4A family genes in tumor immune microenvironment, prognosis, and drug response.

BACKGROUND: NR4A family genes play crucial roles in cancers. However, the role of NR4A family genes in cancers remains paradoxical as they promote or suppress tumorigenesis. OBJECTIVE: We aimed to conduct comprehensive analyses of the association between the expression of NR4A family genes and tumor microenvironment (TME) based on bioinformatics methods. METHODS: We collected RNA-seq data from 33 cancer types and 20 normal tissue sites from the TCGA and GTEx databases. Expression patterns of NR4A family genes and their associations with DNA methylation, miRNA, overall survival, drug responses, and tumor microenvironment were investigated. RESULTS: Significant downregulation of all NR4A family genes was observed in 15 cancer types. DNA promoter methylation and expression of NR4A family genes were negatively correlated in five cancers. The expression of 10 miRNAs targeting NR4A family genes was negatively correlated with the expression of NR4A family genes. High expression of all NR4A family genes was associated with poor prognosis in stomach adenocarcinoma and increased expressions of NR4A2 and NR4A3 were associated with poor prognosis in adrenocortical carcinoma. In addition, we found an elevated expression of NR4A2, which enhances the response to various chemotherapeutic drugs, whereas NR4A3 decreases drug sensitivity. Interestingly, in breast cancer, NR4A3 was significantly associated with C2 (IFN-γ dominant), C3 (inflammatory), and C6 (TGF-ß dominant) immune subtypes and infiltrated immune cell types, implying both oncogenic and tumor-suppressive functions of NR4A3 in breast cancer. CONCLUSION: The NR4A family genes have the potential to serve as a diagnostic, prognostic, and immunological marker of human cancers.

Oxidative stress-induced EGR1 upregulation promotes NR4A3-mediated nucleus pulposus cells apoptosis in intervertebral disc degeneration.

This study aimed to reveal the specific role of early growth response protein 1 (EGR1) and nuclear receptor 4A3 (NR4A3) in nucleus pulposus cells (NPCs) and the related molecular mechanism and to identify a new strategy for treating intervertebral disc degeneration (IVDD). Bioinformatics analysis was used to explore and predict IVDD-related differentially expressed genes, and chromatin immunoprecipitation sequencing (ChIP-seq) revealed NR4A3 as the EGR1 target gene. An in vitro NPC model induced by tributyl hydrogen peroxide (TBHP) and a rat model induced by fibrous ring acupuncture were established. Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemical staining, immunofluorescence staining, and flow cytometry were used to detect the effects of EGR1 and NR4A3 knockdown and overexpression on NPC apoptosis and the expression of extracellular matrix (ECM) anabolism-related proteins. Interactions between EGR1 and NR4A3 were analyzed via ChIP-qPCR and dual luciferase assays. EGR1 and NR4A3 expression levels were significantly higher in severely degenerated discs (SDD) than in mildly degenerated discs (MDD), indicating that these genes are important risk factors in IVDD progression. ChIP-seq and RNA-seq revealed NR4A3 as a direct downstream target of EGR1, and this finding was verified by ChIP-qPCR and dual luciferase reporter experiments. Remarkably, the rescue experiments showed that EGR1 promotes TBHP-induced NPC apoptosis and impairs ECM anabolism, dependent on elevated NR4A3 expression. In summary, the EGR1-NR4A3 axis mediates the progression of NPC apoptosis and ECM impairment and is a potential therapeutic target in IVDD.

Analysis of arsenic-modulated expression of hypothalamic estrogen receptor, thyroid receptor, and peroxisome proliferator-activated receptor gamma mRNA and simultaneous mitochondrial morphology and respiration rates in the mouse.

Arsenic has been identified as an environmental toxicant acting through various mechanisms, including the disruption of endocrine pathways. The present study assessed the ability of a single intraperitoneal injection of arsenic, to modify the mRNA expression levels of estrogen- and thyroid hormone receptors (ERα,ß; TRα,ß) and peroxisome proliferator-activated receptor gamma (PPARγ) in hypothalamic tissue homogenates of prepubertal mice in vivo. Mitochondrial respiration (MRR) was also measured, and the corresponding mitochondrial ultrastructure was analyzed. Results show that ERα,ß, and TRα expression was significantly increased by arsenic, in all concentrations examined. In contrast, TRß and PPARγ remained unaffected after arsenic injection. Arsenic-induced dose-dependent changes in state 4 mitochondrial respiration (St4). Mitochondrial morphology was affected by arsenic in that the 5 mg dose increased the size but decreased the number of mitochondria in agouti-related protein- (AgRP), while increasing the size without affecting the number of mitochondria in pro-opiomelanocortin (POMC) neurons. Arsenic also increased the size of the mitochondrial matrix per host mitochondrion. Complex analysis of dose-dependent response patterns between receptor mRNA, mitochondrial morphology, and mitochondrial respiration in the neuroendocrine hypothalamus suggests that instant arsenic effects on receptor mRNAs may not be directly reflected in St3-4 values, however, mitochondrial dynamics is affected, which predicts more pronounced effects in hypothalamus-regulated homeostatic processes after long-term arsenic exposure.

PON3::LCN1 and HTN3::MSANTD3 Gene Fusions With NR4A3/NR4A2 Expression in Salivary Acinic Cell Carcinoma.

Acinic cell carcinoma of the salivary gland (AciCC) is a low-grade carcinoma characterized by the overexpression of the transcription factor nuclear receptor subfamily 4 group A member 3 (NR4A3). AciCC has been the subject of a few molecular research projects. This study delves into AciCC's molecular landscape to identify additional alterations and explore their clinical implications. RNA sequencing and immunohistochemical staining for markers NR4A3/NR4A2, DOG-1, S100, and mammaglobin were utilized on 41 AciCCs and 11 secretory carcinoma (SC) samples. NR4A3 was evident in 35 AciCCs, while the residual 6 were NR4A3-negative and NR4A2-positive; SC samples were consistently NR4A3-negative. A novel fusion, PON3 exon 1- LCN1 exon 5, was detected in 9/41 (21.9%) AciCCs, exhibiting a classical histologic pattern with serous cell components growing in solid sheets alongside the intercalated duct-like component. Clinical follow-up of 39 patients over a median of 59 months revealed diverse prognostic outcomes: 34 patients exhibited no disease evidence, whereas the remaining 5 experienced poorer prognosis, involving local recurrence, lymph node, and distant metastasis, and disease-associated death, 4 of which harbored the PON3::LCN1 fusion. In addition, the HTN3::MSANTD3 fusion was recurrently identified in 7/41 AciCC cases. SC patients lacked both fusions. Immunohistochemistry uncovered differential expression of DOG-1, S100, and mammaglobin across samples, providing nuanced insights into their roles in AciCC. This study accentuates PON3::LCN1 and HTN3::MSANTD3 fusions as recurrent molecular events in AciCC, offering potential diagnostic and prognostic utility and propelling further research into targeted therapeutic strategies.

Cadmium exposure activates mitophagy through downregulating thyroid hormone receptor/PGC1α signal in preeclampsia.

Gestational cadmium exposure increases the risk of preeclampsia. Placenta mitophagy was activated in preeclampsia. The aim of present study was to explore the mechanism of cadmium-induced mitophagy activation and its association with preeclampsia. Mitophagy markers expression levels were detected by quantitative real-time PCR, Western blot, immunofluorescence and immunochemistry in preeclampsia placenta. JEG3 cells were treated with CdCl2, iopanoic acid (IOP), 3-methyladenine and PGC1α SiRNA to verify mechanism of cadmium-induced mitophagy. Mitophagy marker LC3BII/I and P62 expression were increased and mitochondrial membrane receptor protein TOM20 and FUNDC1 expression were decreased in preeclampsia placenta as compared with that in normotension control. Mitophagy marker LC3BII/I and P62 expression were increased and TOM20 and FUNDC1 expression was decreased in CdCl2-treated JEG3 cells. Meanwhile, mitochondrial biogenesis regulator, PGC1α expression was decreased in preeclampsia and CdCl2-treated JEG3 cells. The expressions of LC3B and P62 were increased and the expressions of TOM20, FUNDC1 and PGC1α were decreased in IOP-treated cell. PGC1α SiRNA transfection led to increased expression of LC3BII/I and P62 and decreased expression of TOM20 and FUNDC1. The expression of sFlt1 was increased in preeclampsia placenta, CdCl2-treated cells, in IOP-treated cells and in PGC1α SiRNA transfected cells. 3-methyladenine treatment protected the increased expression of sFlt1 in CdCl2-treated cells, in IOP-treated cells and in PGC1α SiRNA transfected cells. Meanwhile, co-treatment of cadmium and IOP or PGC1αSiRNA led to a reduce expressions of OPA1, MFN1, MFN2 and FUNDC1 as compared to cadmium-treated, IOP-treated and PGC1α SiRNA-treated cells. These results elucidated that maternal cadmium exposure activated placenta mitophagy through downregulation of thyroid hormone receptor signal mediated decreased expression of PGC1α and was associated with the occurrence of preeclampsia.

Expanding the Spectrum of NR4A3 Fusion-Positive Gynecologic Leiomyosarcomas.

Recurrent gene fusions have been observed in epithelioid and myxoid variants of uterine leiomyosarcoma. PGR::NR4A3 fusions were recently described in a subset of epithelioid leiomyosarcomas exhibiting rhabdoid morphology. In this study, we sought to expand the clinical, morphologic, immunohistochemical, and genetic features of gynecologic leiomyosarcomas harboring NR4A3 rearrangements with PGR and novel fusion partners. We identified 9 gynecologic leiomyosarcomas harboring PGR::NR4A3, CARMN::NR4A3, ACTB::NR4A3, and possible SLCO5A1::NR4A3 fusions by targeted RNA sequencing. Tumors frequently affected premenopausal women, involving the uterine corpus, uterine cervix, or pelvis. All were similarly characterized by lobules of monomorphic epithelioid and/or spindled cells arranged in sheets, cords, trabeculae, and micro- and macrocysts associated with abundant myxoid matrix and hemorrhage, creating labyrinth-like or pulmonary edema-like architecture. Myogenic differentiation with frequent estrogen receptor and progesterone receptor staining and no CD10 expression characterized all tumors. All cases showed high NR4A3 RNA expression levels and NOR1 (NR4A3) nuclear staining similar to salivary gland acinic cell carcinomas and a subset of extraskeletal myxoid chondrosarcomas harboring NR4A3 rearrangements. NOR1 (NR4A3) immunohistochemistry may serve as a useful diagnostic marker of NR4A3 fusion-positive gynecologic leiomyosarcomas.

Truncated variants of thyroid hormone receptor beta display disease-inflicting malfunctioning at cellular level.

Thyroid hormone receptor ß (THRß) is a member of the nuclear receptor superfamily of ligand-modulated transcription factors. Upon ligand binding, THRß sequentially recruits the components of transcriptional machinery to modulate target gene expression. In addition to regulating diverse physiological processes, THRß plays a crucial role in hypothalamus-pituitary-thyroid axis feedback regulation. Anomalies in THRß gene/protein structure are associated with onset of diverse disease states. In this study, we investigated disease-inflicting truncated variants of THRß using in-silico analysis and cell-based assays. We examined the THRß truncated variants on multiple test parameters, including subcellular localization, ligand-receptor interactions, transcriptional functions, interaction with heterodimeric partner RXR, and receptor-chromatin interactions. Moreover, molecular dynamic simulation approaches predicted that shortened THRß-LBD due to point mutations contributes proportionally to the loss of structural integrity and receptor stability. Deviant subcellular localization and compromised transcriptional function were apparent with these truncated variants. Present study shows that 'mitotic bookmarking' property of some THRß variants is also affected. The study highlights that structural and conformational attributes of THRß are necessary for normal receptor functioning, and any deviations may contribute to the underlying cause of the inflicted diseases. We anticipate that insights derived herein may contribute to improved mechanistic understanding to assess disease predisposition.

The ecological function of thyroid hormones.

Thyroid hormones (TH) are central hormonal regulators, orchestrating gene expression and complex biological processes vital for growth and reproduction in variable environments by triggering specific developmental processes in response to external cues. TH serve distinct roles in different species: inducing metamorphosis in amphibians or teleost fishes, governing metabolic processes in mammals, and acting as effectors of seasonality. These multifaceted roles raise questions about the underlying mechanisms of TH action. Recent evidence suggests a shared ecological role of TH across vertebrates, potentially extending to a significant portion of bilaterian species. According to this model, TH ensure that ontogenetic transitions align with environmental conditions, particularly in terms of energy expenditure, helping animals to match their ontogenetic transition with available resources. This alignment spans post-embryonic developmental transitions common to all vertebrates and more subtle adjustments during seasonal changes. The underlying logic of TH function is to synchronize transitions with the environment. This review briefly outlines the fundamental mechanisms of thyroid signalling and shows various ways in which animals use this hormonal system in natural environments. Lastly, we propose a model linking TH signalling, environmental conditions, ontogenetic trajectory and metabolism. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'.

RP11-495P10.1 promotes HCC cell proliferation by regulating reprogramming of glucose metabolism and acetylation of the NR4A3 promoter via the PDK1/PDH axis.

The incidence and related death of hepatocellular carcinoma (HCC) have increased over the past decades. However, the molecular mechanisms underlying HCC pathogenesis are not fully understood. Long noncoding RNA (lncRNA) RP11-495P10.1 has been proven to be closely associated with the progression of prostate cancer, but its role and specific mechanism in HCC are still unknown. Here, we identify that RP11-495P10.1 is highly expressed in HCC tissues and cells and contributes to the proliferation of HCC cells. Moreover, this study demonstrates that RP11-495P10.1 affects the proliferation of HCC by negatively regulating the expression of nuclear receptor subfamily 4 group a member 3 (NR4A3). Glycometabolism reprogramming is one of the main characteristics of tumor cells. In this study, we discover that RP11-495P10.1 regulates glycometabolism reprogramming by changing the expression of pyruvate dehydrogenase kinase 1 (PDK1) and pyruvate dehydrogenase (PDH), thus contributing to the proliferation of HCC cells. Furthermore, knockdown of RP11-495P10.1 increases enrichment of H3K27Ac in the promoter of NR4A3 by promoting the activity of PDH and the production of acetyl-CoA, which leads to the increased transcription of NR4A3. Altogether, RP11-495P10.1 promotes HCC cell proliferation by regulating the reprogramming of glucose metabolism and acetylation of the NR4A3 promoter via the PDK1/PDH axis, which provides an lncRNA-oriented therapeutic strategy for the diagnosis and treatment of HCC.

Thyroid hormone receptor Thra and Thrb knockout differentially affects osteoblast biology and thyroid hormone responsiveness in vitro.

Thyroid hormones (TH) are important modulators of bone remodeling and thus, thyroid diseases, in particular hyperthyroidism, are able to compromise bone quality and fracture resistance. TH actions on bone are mediated by the thyroid hormone receptors (TR) TRα1 and TRß1, encoded by Thra and Thrb, respectively. Skeletal phenotypes of mice lacking Thra (Thra0/0 ) and Thrb (Thrb-/- ) are well-described and suggest that TRα1 is the predominant mediator of TH actions in bone. Considering that bone cells might be affected by systemic TH changes seen in these mutant mice, here we investigated the effects of TR knockout on osteoblasts exclusively at the cellular level. Primary osteoblasts obtained from Thra0/0 , Thrb-/- , and respective wildtype (WT) mice were analyzed regarding their differentiation potential, activity and TH responsiveness in vitro. Thra, but not Thrb knockout promoted differentiation and activity of early, mature and late osteoblasts as compared to respective WT cells. Interestingly, while mineralization capacity and expression of osteoblast marker genes and TH target gene Klf9 was increased by TH in WT and Thra-deficient osteoblasts, Thrb knockout mitigated the responsiveness of osteoblasts to short (48 h) and long term (10 d) TH treatment. Further, we found a low ratio of Rankl, a potent osteoclast stimulator, over osteoprotegerin, an osteoclast inhibitor, in Thrb-deficient osteoblasts and in line, supernatants obtained from Thrb-/- osteoblasts reduced numbers of primary osteoclasts in vitro. In accordance to the increased Rankl/Opg ratio in TH-treated WT osteoblasts only, supernatants from these cells, but not from TH-treated Thrb-/- osteoblasts increased the expression of Trap and Ctsk in osteoclasts, suggesting that osteoclasts are indirectly stimulated by TH via TRß1 in osteoblasts. In conclusion, our study shows that both Thra and Thrb differentially affect activity, differentiation and TH response of osteoblasts in vitro and emphasizes the importance of TRß1 to mediate TH actions in bone.

Overlaps Between CDS Regions of Protein-Coding Genes in the Human Genome: A Case Study on the NR1D1-THRA Gene Pair.

For several decades, it has been known that a substantial number of genes within human DNA exhibit overlap; however, the biological and evolutionary significance of these overlaps remain poorly understood. This study focused on investigating specific instances of overlap where the overlapping DNA region encompasses the coding DNA sequences (CDSs) of protein-coding genes. The results revealed that proteins encoded by overlapping CDSs exhibit greater disorder than those from nonoverlapping CDSs. Additionally, these DNA regions were identified as GC-rich. This could be partially attributed to the absence of stop codons from two distinct reading frames rather than one. Furthermore, these regions were found to harbour fewer single-nucleotide polymorphism (SNP) sites, possibly due to constraints arising from the overlapping state where mutations could affect two genes simultaneously.While elucidating these properties, the NR1D1-THRA gene pair emerged as an exceptional case with highly structured proteins and a distinctly conserved sequence across eutherian mammals. Both NR1D1 and THRA are nuclear receptors lacking a ligand-binding domain at their C-terminus, which is the region where these gene pairs overlap. The NR1D1 gene is involved in the regulation of circadian rhythm, while the THRA gene encodes a thyroid hormone receptor, and both play crucial roles in various physiological processes. This study suggests that, in addition to their well-established functions, the specifically overlapping CDS regions of these genes may encode protein segments with additional, yet undiscovered, biological roles.

Essential and subtype-dependent function of thyroid hormone receptors during Xenopus metamorphosis.

Thyroid hormone (T3) plays critical roles in organ metabolism and development in vertebrates. Anuran metamorphosis is perhaps the most dramatic and best studied developmental process controlled by T3. Many changes in different organs/tissues during anuran metamorphosis resemble the maturation/remodeling of the corresponding organs/tissues during mammalian postembryonic development. The plasma T3 level peaks during both anuran metamorphosis and mammalian postembryonic development. T3 exerts its developmental function through transcriptional regulation via T3 receptors (TRs). Studies on the metamorphosis of two highly related anurans, pseudo-tetraploid Xenopus laevis and diploid Xenopus tropicalis, have led to a dual function model for TRs during development. This has been supported by strong molecular and genetic evidence. Here we review some of the evidence with a focus on more recent gene knockout studies in Xenopus tropicalis. These studies have not only supported the model but also revealed novel and TR subtype-specific roles during Xenopus development, particularly a critical role of TRα in controlling developmental timing and rate.

A Historical Reflection on Scientific Advances in Understanding Thyroid Hormone Action.

Background: Thyroid hormone (TH) has actions in every tissue of the body and is essential for normal development, as well as having important actions in the adult. The earliest markers of TH action that were identified and monitored clinically, even before TH could be measured in serum, included oxygen consumption, basal metabolic rate, serum cholesterol, and deep tendon reflex time. Cellular, rodent, amphibian, zebrafish, and human models have been used to study TH action. Summary: Early studies of the mechanism of TH action focused on saturable-specific triiodothyronine (T3) nuclear binding and direct actions of T3 that altered protein expression. Additional effects of TH were recognized on mitochondria, stimulation of ion transport, especially the sodium potassium ATPase, augmentation of adrenergic signaling, role as a neurotransmitter, and direct plasma membrane effects. The cloning of the thyroid hormone receptor (THR) genes in 1986 and report of the THR crystal structure in 1995 produced rapid progress in understanding the mechanism of TH nuclear action, as well as the development of modified THR ligands. These findings revealed nuances of TH signaling, including the role of nuclear receptor coactivators and corepressors, repression of positively stimulated genes by the unliganded receptor, THR isoform-specific actions of TRα (THRA) and TRß (THRB), and THR binding DNA as a heterodimer with retinoid-x-receptor (RXR) for genes positively regulated by TH. The identification of genetic disorders of TH transport and signaling, especially Resistance to Thyroid Hormone (RTH) and monocarboxylate transporter 8 (Mct8) defects, has been highly informative with respect to the mechanism of TH action. Conclusions: The impact of THR isoform, post-translational modifications, receptor cofactors, DNA response element, and selective TH tissue uptake, on TH action, have clinical implications for diagnosing and treating thyroid disease. Additionally, these findings have led to the development of novel TH and TH analogue therapies for metabolic, neurological, and cardiovascular diseases.

The role of transducin ß-like 1 X-linked receptor 1 (TBL1XR1) in thyroid hormone metabolism and action in mice.

Transducin ß-like 1 X-linked receptor 1 (TBL1XR1) is a WD40 repeat-containing protein and part of the corepressor complex SMRT/NCoR that binds to the thyroid hormone receptor (TR). We recently described a mutation in TBL1XR1 in patients with Pierpont syndrome. A mouse model bearing this Tbl1xr1 mutation (Tbl1xr1Y446C/Y446C ) displays several aspects of the Pierpont phenotype. Although serum thyroid hormone (TH) concentrations were unremarkable in these mice, tissue TH action might be affected due to the role of TBL1XR1 in the SMRT/NCoR corepressor complex. The aim of the present study was to evaluate tissue TH metabolism and action in a variety of tissues of Tbl1xr1Y446C/Y446C mice. We studied the expression of genes involved in TH metabolism and action in tissues of naïve Tbl1xr1Y446C/Y446C mice and wild type (WT) mice. In addition, we measured deiodinase activity in liver (Dio1 and Dio3), kidney (Dio1 and Dio3) and BAT (Dio2). No striking differences were observed in the liver, hypothalamus, muscle and BAT between Tbl1xr1Y446C/Y446C and WT mice. Pituitary TRα1 mRNA expression was lower in Tbl1xr1Y446C/Y446C mice compared to WT, while the mRNA expression of Tshß and the positively T3-regulated gene Nmb were significantly increased in mutant mice. Interestingly, Mct8 expression was markedly higher in WAT and kidney of mutants, resulting in (subtle) changes in T3-regulated gene expression in both WAT and kidney. In conclusion, mice harboring a mutation in TBL1XR1 display minor changes in cellular TH metabolism and action. TH transport via MCT8 might be affected as the expression is increased in WAT and kidney. The mechanisms involved need to be clarified.

EWSR1::NR4A3 gene fusion in a cutaneous atypical myoepithelial neoplasm.

Myoepithelial neoplasms of the skin and soft tissue are rare and share histopathologic features with their salivary gland counterpart. We present a case of an atypical myoepithelial neoplasm from the back of a 72-year-old female. This lesion harbored an EWSR1::NR4A3 gene fusion, a genetic signature characteristically seen in extraskeletal myxoid chondrosarcoma. To our knowledge, this is a unique case of an atypical cutaneous myoepithelial neoplasm harboring EWSR1::NR4A3 fusion.

Stem cell development involves divergent thyroid hormone receptor subtype expression and epigenetic modifications in the amphibian intestine during metamorphosis.

In the amphibian intestine during metamorphosis, most of the larval epithelial cells undergo apoptosis, while a small number of the epithelial cells dedifferentiate into stem cells (SCs). The SCs actively proliferate and then newly generate the adult epithelium analogous to the mammalian counterpart, which is continuously renewed from the SCs throughout adulthood. This larval-to-adult intestinal remodeling can be experimentally induced by thyroid hormone (TH) through interacting with the surrounding connective tissue that develops as the stem cell niche. Thus, the amphibian intestine provides us a valuable opportunity to study how the SCs and their niche are formed during development. To clarify the TH-induced and evolutionally conserved mechanism of SC development at the molecular level, numerous TH response genes have been identified in the Xenopus laevis intestine over the last three decades and extensively analyzed for their expression and function by using wild-type and transgenic Xenopus tadpoles. Interestingly, accumulating evidence indicates that thyroid hormone receptor (TR) epigenetically regulates the expression of TH response genes involved in the remodeling. In this review, we highlight recent progress in the understanding of SC development, focusing on epigenetic gene regulation by TH/TR signaling in the X. laevis intestine. We here propose that two subtypes of TRs, TRα and TRß, play distinct roles in the intestinal SC development via different histone modifications in different cell types.

Extraskeletal Myxoid Chondrosarcomas: The Uncommon Clinicopathologic Manifestations and Significance of TAF15::NR4A3 Fusion.

Extraskeletal myxoid chondrosarcoma (EMC) is an ultrarare sarcoma typically exhibiting myxoid/reticular histology and NR4A3 translocation. However, morphologic variants and the relevance of non-EWSR1::NR4A3 fusions remain underexplored. Three challenging pan-Trk-expressing cases, featuring cellular to solid histology, were subjected to RNA exome sequencing (RES), unveiling different NR4A3-associated fusions. Alongside RES-analyzed cases, fluorescence in situ hybridization was performed to confirm 58 EMCs, with 48 available for pan-Trk immunostaining and KIT sequencing. Except for 1 (2%) NR4A3-rearranged EMC without identifiable partners, 46 (79%), 9 (16%), and 2 (3%) cases harbored EWSR1::NR4A3, TAF15::NR4A3, and TCF12::NR4A3 fusions, respectively. Five EWSR1::NR4A3-positive EMCs occurred in the subcutis (3) and bone (2). Besides 43 classical cases, there were 8 cellular, 4 rhabdoid/anaplastic, 2 solid, and 1 mixed tumor-like variants. Tumor cells were oval/spindle to pleomorphic and formed loose myxoid/reticular to compact sheet-like or fascicular patterns, imparting broad diagnostic considerations. RES showed upregulation of NTRK2/3, KIT, and INSM1. Moderate-to-strong immunoreactivities of pan-Trk, CD117, and INSM1 were present in 35.4%, 52.6%, and 54.6% of EMCs, respectively. KIT p. E554K mutation was detected in 2/48 cases. TAF15::NR4A3 was significantly associated with size >10 cm (78%, P = .025). Size >10 cm, moderate-to-severe nuclear pleomorphism, metastasis at presentation, TAF15::NR4A3 fusion, and the administration of chemotherapy portended shorter univariate disease-specific survival, whereas only size >10 cm (P = .004) and metastasis at presentation (P = .032) remained prognostically independent. Conclusively, EMC may manifest superficial or osseous lesions harboring EWSR1::NR4A3, underrecognized solid or anaplastic histology, and pan-Trk expression, posing tremendous challenges. Most TAF15::NR4A3-positive cases were >10 cm in size, ie, a crucial independent prognosticator, whereas pathogenic KIT mutation rarely occurred.