International Union of Basic and Clinical Pharmacology CXIII: Nuclear Receptor Superfamily-Update 2023.

The NR superfamily comprises 48 transcription factors in humans that control a plethora of gene network programs involved in a wide range of physiologic processes. This review will summarize and discuss recent progress in NR biology and drug development derived from integrating various approaches, including biophysical techniques, structural studies, and translational investigation. We also highlight how defective NR signaling results in various diseases and disorders and how NRs can be targeted for therapeutic intervention via modulation via binding to synthetic lipophilic ligands. Furthermore, we also review recent studies that improved our understanding of NR structure and signaling. SIGNIFICANCE STATEMENT: Nuclear receptors (NRs) are ligand-regulated transcription factors that are critical regulators of myriad physiological processes. NRs serve as receptors for an array of drugs, and in this review, we provide an update on recent research into the roles of these drug targets.

Multiple mechanisms regulate H3 acetylation of enhancers in response to thyroid hormone.

Hormone-dependent activation of enhancers includes histone hyperacetylation and mediator recruitment. Histone hyperacetylation is mostly explained by a bimodal switch model, where histone deacetylases (HDACs) disassociate from chromatin, and histone acetyl transferases (HATs) are recruited. This model builds on decades of research on steroid receptor regulation of transcription. Yet, the general concept of the bimodal switch model has not been rigorously tested genome wide. We have used a genomics approach to study enhancer hyperacetylation by the thyroid hormone receptor (TR), described to operate as a bimodal switch. H3 acetylation, HAT and HDAC ChIP-seq analyses of livers from hypo- and hyperthyroid wildtype, TR deficient and NCOR1 disrupted mice reveal three types of thyroid hormone (T3)-regulated enhancers. One subset of enhancers is bound by HDAC3-NCOR1 in the absence of hormone and constitutively occupy TR and HATs irrespective of T3 levels, suggesting a poised enhancer state in absence of hormone. In presence of T3, HDAC3-NCOR1 dissociates from these enhancers leading to histone hyperacetylation, suggesting a histone acetylation rheostat function of HDACs at poised enhancers. Another subset of enhancers, not occupied by HDACs, is hyperacetylated in a T3-dependent manner, where TR is recruited to chromatin together with HATs. Lastly, a subset of enhancers, is not occupied directly by TR yet requires TR for histone hyperacetylation. This indirect enhancer activation involves co-association with TR bound enhancers within super-enhancers or topological associated domains. Collectively, this demonstrates various mechanisms controlling hormone-dependent transcription and adds significant details to the otherwise simple bimodal switch model.

Thyroid Hormone Signaling and the Liver.

Thyroid hormone (TH) plays a critical role in maintaining metabolic homeostasis throughout life. It is well known that the liver and thyroid are intimately linked, with TH playing important roles in de novo lipogenesis, beta-oxidation (fatty acid oxidation), cholesterol metabolism, and carbohydrate metabolism. Indeed, patients with hypothyroidism have abnormal lipid panels with higher levels of low-density lipoprotein levels, triglycerides (triacylglycerol; TAG), and apolipoprotein B levels. Even in euthyroid patients, lower serum-free thyroxine levels are associated with higher total cholesterol levels, LDL, and TAG levels. In addition to abnormal serum lipids, the risk of nonalcoholic fatty liver disease (NAFLD) increases with lower free thyroxine levels. As free thyroxine rises, the risk of NAFLD is reduced. This has led to numerous animal studies and clinical trials investigating TH analogs and TH receptor agonists as potential therapies for NAFLD and hyperlipidemia. Thus, TH plays an important role in maintaining hepatic homeostasis, and this continues to be an important area of study. A review of TH action and TH actions on the liver will be presented here.

A joint industry-sponsored data monitoring committee model for observational, retrospective drug safety studies in the real-world setting.

PURPOSE: To share better practice in establishing data monitoring committees (DMCs) for observational, retrospective safety studies with joint-industry sponsorship. METHODS: A DMC model was created to monitor data from an observational, retrospective, post-authorization safety study investigating risk of medullary thyroid cancer in patients treated with long-acting glucagon-like peptide-1 receptor agonists (LA GLP-1RAs) (NCT01511393). Sponsors reviewed regulatory guidelines, best practice and sponsors' standard operation procedures on DMCs. Discussions were held within the four-member consortium, assessing applicability to observational, retrospective, real-world studies. A DMC charter was drafted based on a sponsor-proposed, adapted DMC model. Thereafter, a kick-off meeting between sponsors and DMC members was held to receive DMC input and finalize the charter. RESULTS: Due to this study's observational, retrospective nature, assuring participant safety - central for traditional explanatory clinical trial models - was not applicable to our DMC model. The overall strategy and key indication for our real-world model included preserving study integrity and credibility. Therefore, DMC member independence and their contribution of expert knowledge were essential. To ensure between-sponsor data confidentiality, all study committees/corporations and sponsors, besides the DMC, received blinded data only (adapted to refer to data blinding that revealed the specific marketed LA GLP-1RA/sponsor). Communication and blinding/unblinding of these data were facilitated by the contract research organization, which also provided crucial operational oversight. CONCLUSIONS: To our knowledge, we have established the first DMC model for joint industry-sponsored, observational, retrospective safety studies. This model could serve as a precedent for others performing similar post-marketing, joint industry-sponsored pharmacovigilance activities.

Thyroid and Cardiovascular Disease: Research Agenda for Enhancing Knowledge, Prevention, and Treatment.

Thyroid hormones have long been known to have a range of effects on the cardiovascular system. However, significant knowledge gaps exist concerning the precise molecular and biochemical mechanisms governing these effects and the optimal strategies for management of abnormalities in thyroid function in patients with and without preexisting cardiovascular disease. In September 2017, the National Heart, Lung, and Blood Institute convened a Working Group with the goal of developing priorities for future scientific research relating thyroid dysfunction to the progression of cardiovascular disease. The Working Group reviewed and discussed the roles of normal thyroid physiology, the consequences of thyroid dysfunction, and the effects of therapy in 3 cardiovascular areas: cardiac electrophysiology and arrhythmias, the vasculature and atherosclerosis, and the myocardium and heart failure. This report describes the current state of the field, outlines barriers and challenges to progress, and proposes research opportunities to advance the field, including strategies for leveraging novel approaches using omics and big data. The Working Group recommended research in 3 broad areas: (1) investigation into the fundamental biology relating thyroid dysfunction to the development of cardiovascular disease and into the identification of novel biomarkers of thyroid hormone action in cardiovascular tissues; (2) studies that define subgroups of patients with thyroid dysfunction amenable to specific preventive strategies and interventional therapies related to cardiovascular disease; and (3) clinical trials focused on improvement in cardiovascular performance and cardiovascular outcomes through treatment with thyroid hormone or thyromimetic drugs.

Pluripotent stem cell differentiation reveals distinct developmental pathways regulating lung- versus thyroid-lineage specification.

The in vitro-directed differentiation of pluripotent stem cells (PSCs) through stimulation of developmental signaling pathways can generate mature somatic cell types for basic laboratory studies or regenerative therapies. However, there has been significant uncertainty regarding a method to separately derive lung versus thyroid epithelial lineages, as these two cell types each originate from Nkx2-1+ foregut progenitors and the minimal pathways claimed to regulate their distinct lineage specification in vivo or in vitro have varied in previous reports. Here, we employ PSCs to identify the key minimal signaling pathways (Wnt+BMP versus BMP+FGF) that regulate distinct lung- versus thyroid-lineage specification, respectively, from foregut endoderm. In contrast to most previous reports, these minimal pathways appear to be evolutionarily conserved between mice and humans, and FGF signaling, although required for thyroid specification, unexpectedly appears to be dispensable for lung specification. Once specified, distinct Nkx2-1+ lung or thyroid progenitor pools can now be independently derived for functional 3D culture maturation, basic developmental studies or future regenerative therapies.

NCoR1-independent mechanism plays a role in the action of the unliganded thyroid hormone receptor.

Nuclear receptor corepressor 1 (NCoR1) is considered to be the major corepressor that mediates ligand-independent actions of the thyroid hormone receptor (TR) during development and in hypothyroidism. We tested this by expressing a hypomorphic NCoR1 allele (NCoR1ΔID), which cannot interact with the TR, in Pax8-KO mice, which make no thyroid hormone. Surprisingly, abrogation of NCoR1 function did not reverse the ligand-independent action of the TR on many gene targets and did not fully rescue the high mortality rate due to congenital hypothyroidism in these mice. To further examine NCoR1's role in repression by the unliganded TR, we deleted NCoR1 in the livers of euthyroid and hypothyroid mice and examined the effects on gene expression and enhancer activity measured by histone 3 lysine 27 (H3K27) acetylation. Even in the absence of NCoR1 function, we observed strong repression of more than 43% of positive T3 (3,3',5-triiodothyronine) targets in hypothyroid mice. Regulation of approximately half of those genes correlated with decreased H3K27 acetylation, and nearly 80% of these regions with affected H3K27 acetylation contained a bona fide TRß1-binding site. Moreover, using liver-specific TRß1-KO mice, we demonstrate that hypothyroidism-associated changes in gene expression and histone acetylation require TRß1. Thus, many of the genomic changes mediated by the TR in hypothyroidism are independent of NCoR1, suggesting a role for additional signaling modulators in hypothyroidism.

Fructose and glucose can regulate mammalian target of rapamycin complex 1 and lipogenic gene expression via distinct pathways.

Although calorically equivalent to glucose, fructose appears to be more lipogenic, promoting dyslipidemia, fatty liver disease, cardiovascular disease, and diabetes. To better understand how fructose induces lipogenesis, we compared the effects of fructose and glucose on mammalian target of rapamycin complex 1 (mTORC1), which appeared to have both positive and negative effects on lipogenic gene expression. We found that fructose acutely and transiently suppressed mTORC1 signaling in vitro and in vivo The constitutive activation of mTORC1 reduced hepatic lipogenic gene expression and produced hypotriglyceridemia after 1 week of fructose feeding. In contrast, glucose did not suppress mTORC1, and the constitutive activation of mTORC1 failed to suppress plasma triglycerides after 1 week of glucose feeding. Thus, these data reveal fundamental differences in the signaling pathways used by fructose and glucose to regulate lipid metabolism.

Hepatic FOXO1 Target Genes Are Co-regulated by Thyroid Hormone via RICTOR Protein Deacetylation and MTORC2-AKT Protein Inhibition.

MTORC2-AKT is a key regulator of carbohydrate metabolism and insulin signaling due to its effects on FOXO1 phosphorylation. Interestingly, both FOXO1 and thyroid hormone (TH) have similar effects on carbohydrate and energy metabolism as well as overlapping transcriptional regulation of many target genes. Currently, little is known about the regulation of MTORC2-AKT or FOXO1 by TH. Accordingly, we performed hepatic transcriptome profiling in mice after FOXO1 knockdown in the absence or presence of TH, and we compared these results with hepatic FOXO1 and THRB1 (TRß1) ChIP-Seq data. We identified a subset of TH-stimulated FOXO1 target genes that required co-regulation by FOXO1 and TH. TH activation of FOXO1 was directly linked to an increase in SIRT1-MTORC2 interaction and RICTOR deacetylation. This, in turn, led to decreased AKT and FOXO1 phosphorylation. Moreover, TH increased FOXO1 nuclear localization, DNA binding, and target gene transcription by reducing AKT-dependent FOXO1 phosphorylation in a THRB1-dependent manner. These events were associated with TH-mediated oxidative phosphorylation and NAD(+) production and suggested that downstream metabolic effects by TH can post-translationally activate other transcription factors. Our results showed that RICTOR/MTORC2-AKT can integrate convergent hormonal and metabolic signals to provide coordinated and sensitive regulation of hepatic FOXO1-target gene expression.

Nuclear receptor corepressor (NCOR1) regulates in vivo actions of a mutated thyroid hormone receptor α.

Genetic evidence from patients with mutations of the thyroid hormone receptor α gene (THRA) indicates that the dominant negative activity of mutants underlies the pathological manifestations. However, the molecular mechanisms by which TRα1 mutants exert dominant negative activity in vivo are not clear. We tested the hypothesis that the severe hypothyroidism in patients with THRA mutations is due to an inability of TRα1 mutants to properly release the nuclear corepressors (NCORs), thereby inhibiting thyroid hormone-mediated transcription activity. We crossed Thra1(PV) mice, expressing a dominant negative TRα1 mutant (TRα1PV), with mice expressing a mutant Ncor1 allele (Ncor1(ΔID) mice) that cannot recruit the TR or PV mutant. TRα1PV shares the same C-terminal mutated sequences as those of patients with frameshift mutations of the THRA gene. Remarkably, NCOR1ΔID ameliorated abnormalities in the thyroid-pituitary axis of Thra1(PV/+) mice. The severe retarded growth, infertility, and delayed bone development were partially reverted in Thra1(PV/+) mice expressing NCOR1ΔID. The impaired adipogenesis was partially corrected by de-repression of peroxisome-proliferator activated receptor γ and CCAAT/enhancer-binding protein α gene, due to the inability of TRα1PV to recruit NCOR1ΔID to form a repressor complex. Thus, the aberrant recruitment of NCOR1 by TRα1 mutants could lead to clinical hypothyroidism in humans. Therefore, therapies aimed at the TRα1-NCOR1 interaction or its downstream actions could be tested as potential targets in treating TRα1 mutant-mediated hypothyroidism in patients.

Novel mechanism of positive versus negative regulation by thyroid hormone receptor ß1 (TRß1) identified by genome-wide profiling of binding sites in mouse liver.

Triiodothyronine (T3) regulates key metabolic processes in the liver through the thyroid hormone receptor, TRß1. However, the number of known target genes directly regulated by TRß1 is limited, and the mechanisms by which positive and especially negative transcriptional regulation occur are not well understood. To characterize the TRß1 cistrome in vivo, we expressed a biotinylated TRß1 in hypo- and hyperthyroid mouse livers, used ChIP-seq to identify genomic TRß1 targets, and correlated these data with gene expression changes. As with other nuclear receptors, the majority of TRß1 binding sites were not in proximal promoters but in the gene body of known genes. Remarkably, T3 can dictate changes in TRß1 binding, with strong correlation to T3-induced gene expression changes, suggesting that differential TRß1 binding regulates transcriptional outcome. Additionally, DR-4 and DR-0 motifs were significantly enriched at binding sites where T3 induced an increase or decrease in TRß1 binding, respectively, leading to either positive or negative regulation by T3. Taken together, the results of this study provide new insights into the mechanisms of transcriptional regulation by TRß1 in vivo.

The in vivo role of nuclear receptor corepressors in thyroid hormone action.

BACKGROUND: The thyroid hormone receptor (TR) isoforms interact with a variety of coregulators depending upon the availability of T3 to mediate their transcriptional effect. Classically, in the absence of ligand, the TRs recruit the nuclear corepressors, NCoR and SMRT, to mediate transcriptional repression on positively regulated TR target genes. However, new insight into the roles of NCoR and SMRT using in vivo models have better defined the role of nuclear corepressors both in the absence and presence of T3. SCOPE OF REVIEW: This review will place the variety of in vivo nuclear corepressor mouse models developed to date in context of thyroid hormone action. Based on these models, we will also discuss how corepressor availability together with the levels of endogenous nuclear receptor ligands including T3 controls multiple signaling pathways. MAJOR CONCLUSIONS: Nuclear corepressors mediate repression of positive TR targets in the absence of T3in vivo. Even more importantly they attenuate activation of these targets at the normal physiological levels of ligands by TR and other nuclear receptors. While the role of corepressors in the regulation of negative TR targets and HPT axis remains poorly understood, lack of corepressor recruitment to TR in the animals leads to a compensatory change in the set point of HPT axis that allows to balance the increased sensitivity to T3 action in other tissues. GENERAL SIGNIFICANCE: Available data indicate that targeting specific interactions between corepressors and TR or other nuclear receptors presents a new therapeutic strategy for endocrine and metabolic disorders. This article is part of a Special Issue entitled Thyroid hormone signalling.

Resistance to thyroid hormone is modulated in vivo by the nuclear receptor corepressor (NCOR1).

Mutations in the ligand-binding domain of the thyroid hormone receptor ß (TRß) lead to resistance to thyroid hormone (RTH). These TRß mutants function in a dominant-negative fashion to interfere with the transcription activity of wild-type thyroid hormone receptors (TRs), leading to dysregulation of the pituitary-thyroid axis and resistance in peripheral tissues. The molecular mechanism by which TRß mutants cause RTH has been postulated to be an inability of the mutants to properly release the nuclear corepressors (NCORs), thereby inhibiting thyroid hormone (TH)-mediated transcription activity. To test this hypothesis in vivo, we crossed Thrb(PV) mice (a model of RTH) expressing a human TRß mutant (PV) with mice expressing a mutant Ncor1 allele (Ncor1(ΔID) mice) that cannot recruit a TR or a PV mutant. Remarkably, in the presence of NCOR1ΔID, the abnormally elevated thyroid-stimulating hormone and TH levels found in Thrb(PV) mice were modestly but significantly corrected. Furthermore, thyroid hyperplasia, weight loss, and other hallmarks of RTH were also partially reverted in mice expressing NCOR1ΔID. Taken together, these data suggest that the aberrant recruitment of NCOR1 by RTH TRß mutants leads to clinical RTH in humans. The present study suggests that therapies aimed at the TR-NCOR1 interaction or its downstream actions could be tested as potential targets in treating RTH.

The role of WD40 repeat-containing proteins in endocrine (dys)function.

WD40 repeat-containing proteins play a key role in many cellular functions including signal transduction, protein degradation, and apoptosis. The WD40 domain is highly conserved, and its typical structure is a ß-propeller consisting of 4-8 blades which probably serves as a scaffold for protein-protein interaction. Some WD40 repeat-containing proteins form part of the corepressor complex of nuclear hormone receptors, a family of ligand-dependent transcription factors that play a central role in the regulation of gene transcription. This explains their involvement in endocrine physiology and pathology. In the present review, we first touch upon the structure of WD40 repeat-containing proteins. Next, we describe our current understanding of the role of WD40 domain-containing proteins in nuclear receptor signaling, e.g., as corepressor or coactivator. In the final part of this review, we focus on WD40 domain-containing proteins that are associated with endocrine pathologies. These pathologies vary from isolated dysfunction of one endocrine axis, e.g., congenital isolated central hypothyroidism, to more complex congenital syndromes comprising endocrine phenotypes, such as the Triple-A syndrome.

Regulation of Thyroid Hormone Levels by Hypothalamic Thyrotropin-Releasing Hormone Neurons.

Background: Thyrotropin-releasing hormone (TRH) neurons in the paraventricular nucleus of the hypothalamus (PVN) have been identified as direct regulators of thyrotropin (TSH) and thyroid hormone (TH) levels. They play a significant role in context of negative feedback by TH at the level of TRH gene expression and during fasting when TH levels fall due, in part, to suppression of TRH gene expression. Methods: To test these functions directly for the first time, we used a chemogenetic approach and activated PVN TRH neurons in both fed and fasted mice. Next, to demonstrate the signals that regulate the fasting response in TRH neurons, we activated or inhibited agouti-related protein (AgRP)/neuropeptide Y (NPY) neurons in the arcuate nucleus of the hypothalamus of fed or fasted mice, respectively. To determine if the same TRH neurons responsive to melanocortin signaling mediate negative feedback by TH, we disrupted the thyroid hormone receptor beta (TRß) in all melanocortin 4 receptor (MC4R) neurons in the PVN. Results: Activation of TRH neurons led to increased TSH and TH levels within 2 hours demonstrating the specific role of PVN TRH neurons in the regulation of the hypothalamic-pituitary-thyroid (HPT) axis. Moreover, activation of PVN TRH neurons prevented the fall in TH levels in fasting mice. Stimulation of AgRP/NPY neurons led to a fall in TH levels despite increasing feeding. Inhibition of these same neurons prevented the fall in TH levels during a fast presumably via their ability to directly regulate PVN TRH neurons via, in part, the MC4R. Surprisingly, TH-mediated feedback was not impaired in mice lacking TRß in MC4R neurons. Conclusions: TRH neurons are major regulators of the HPT axis and the fasting-induced suppression of TH levels. The latter relies, at least in part, on the activation of AgRP/NPY neurons in the arcuate nucleus. Interestingly, present data do not support an important role for TRß signaling in regulating MC4R neurons in the PVN. Thus, it remains possible that different subsets of TRH neurons in the PVN mediate responses to energy balance and to TH feedback.

Thyroid hormone-regulated chromatin landscape and transcriptional sensitivity of the pituitary gland.

Thyroid hormone (3,5,3'-triiodothyronine, T3) is a key regulator of pituitary gland function. The response to T3 is thought to hinge crucially on interactions of nuclear T3 receptors with enhancers but these sites in pituitary chromatin remain surprisingly obscure. Here, we investigate genome-wide receptor binding in mice using tagged endogenous thyroid hormone receptor ß (TRß) and analyze T3-regulated open chromatin using an anterior pituitary-specific Cre driver (Thrbb2Cre). Strikingly, T3 regulates histone modifications and chromatin opening primarily at sites that maintain TRß binding regardless of T3 levels rather than at sites where T3 abolishes or induces de novo binding. These sites associate more frequently with T3-activated than T3-suppressed genes. TRß-deficiency blunts T3-regulated gene expression, indicating that TRß confers transcriptional sensitivity. We propose a model of gene activation in which poised receptor-enhancer complexes facilitate adjustable responses to T3 fluctuations, suggesting a genomic basis for T3-dependent pituitary function or pituitary dysfunction in thyroid disorders.

Lack of cAMP-response element-binding protein 1 in the hypothalamus causes obesity.

The melanocortin system in the hypothalamus controls food intake and energy expenditure. Its disruption causes severe obesity in mice and humans. cAMP-response element-binding protein 1 (CREB1) has been postulated to play an important role downstream of the melanocortin-4 receptor (MC4R), but this hypothesis has never been confirmed in vivo. To test this, we generated mice that lack CREB1 in SIM1-expressing neurons, of the paraventricular nucleus (PVN), which are known to be MC4R-positive. Interestingly, CREB1(ΔSIM1) mice developed obesity as a result of decreased energy expenditure and impairment in maintaining their core body temperature and not because of hyperphagia, defining a new role for CREB1 in the PVN. In addition, the lack of CREB1 in the PVN caused a reduction in vasopressin expression but did not affect adrenal or thyroid function. Surprisingly, MC4R function tested pharmacologically was normal in CREB1(ΔSIM1) mice, suggesting that CREB1 is not required for intact MC4R signaling. Thus CREB1 may affect other pathways that are implicated in the regulation of body weight.

Reducing systemic hypermetabolism by inducing hypothyroidism does not prolong survival in the SOD1-G93A mouse.

ALS is commonly associated with a hypermetabolic state. In this study, we assess whether inhibition of this hypermetabolism via the induction of hypothyroidism can forestall disease onset and prolong life in the SOD1-G93A mouse. We treated a cohort of 16 SOD1-G93A mice with methimazole, a potent inhibitor of thyroid hormone synthesis and followed a second group of 23 untreated littermate control animals from approximately five weeks of age onward. Total thyroxine (T4) levels, weights, and rectal temperatures were obtained on a regular basis and animals were sacrificed when they were no longer able to feed themselves. Results revealed that T4 levels were effectively suppressed within two weeks of drug initiation. However, there was no significant difference between the two groups either in terms of clinical disease onset (120.1±9.3 days for treated animals and 116.7±6.3 days for untreated animals) or in terms of survival (131.4±11.7 days for treated animals and 134.0±10.0 days for untreated animals). A correlation analysis between mean T4 levels for each animal versus survival showed that, contrary to our hypothesis, higher T4 levels correlated with longer survival. In conclusion, these studies show that drug-induced hypothyroidism does not alter the disease course in the SOD1-G93A ALS mouse.

Impact of the KL2/Catalyst Medical Research Investigator Training (CMeRIT) Program on the careers of early-stage clinical and translational investigators.

The Harvard Catalyst KL2/CMeRIT program is a 2-year mentored institutional career award that includes KL2 grants funded by National Institutes of Health (NIH) and CMeRIT grants funded by Harvard Catalyst nonfederal funds. The purpose of this study was to compare outcomes for early-stage investigators funded by the KL2/CMeRIT program to a group of applicants who were not chosen for support to assess the potential impact of the program on early career outcomes. Career data, including academic promotions, subsequent grant funding, and publication rates, from both successful and unsuccessful 2008-2018 KL2/CMeRIT applicants were compiled throughout the year 2020. Data were obtained directly through outreach to both groups and through assessment of online resources. The cohort comprised 487 individuals, 109 awardees, and 378 nonawardees. Awardees were more likely to be subsequently involved in clinical and translational research than nonawardees (92% vs 75%, p < 0.001). A higher proportion of awardees also had achieved academic promotion (81% vs 69%, p = 0.016) and subsequent NIH funding (72% vs 58%, p = 0.047), while there was no difference in publication rates (p = 0.555). Participants in the Harvard Catalyst KL2/CMeRIT program demonstrate greater early career success than nonparticipants though the nonparticipants also fared relatively well.

Nuclear corepressor SMRT acts as a strong regulator of both ß-oxidation and suppressor of fibrosis in the differentiation process of mouse skeletal muscle cells.

BACKGROUND: Silencing Mediator of Retinoid and Thyroid hormone receptors (SMRT; NCoR2) is a transcriptional corepressor (CoR) which has been recognized as an important player in the regulation of hepatic lipogenesis and in somatic development in mouse embryo. SMRT protein is also widely expressed in mouse connective tissues, for example adipocytes and muscle. We recently reported that mice with global deletion of SMRT develop significant obesity and muscle wasting which are independent from thyroid hormone (TH) signaling and thermogenesis. However, the tissue specific role of SMRT in skeletal muscle is still not clear. METHODS: To clarify role of SMRT in muscle differentiation, we made myogenic C2C12 clones which lack SMRT protein (C2C12-SKO) by using CRISPR-Cas9. Wild-type C2C12 (C2C12-WT) and C2C12-SKO cells were cultured in differentiation medium, and the resulting gene and protein profiles were compared between the two cell lines both before and after differentiation. We also analyzed muscle tissues which were dissected from whole body SMRT knockout (KO) mice and their controls. RESULTS: We found significant up-regulation of muscle specific ß-oxidation markers; Peroxisome proliferator-activated receptor δ (PPARδ) and PPARγ coactivator-1α (PGC-1α) in the C2C12-SKO cells, suggesting that the cells had a similar gene profile to what is found in exercised rodent skeletal muscle. On the other hand, confocal microscopic analysis showed the significant loss of myotubes in C2C12-SKO cells similar to the morphology found in immature myoblasts. Proteomics analysis also confirmed that the C2C12-SKO cells had higher expression of markers of fibrosis (ex. Collagen1A1; COL1A1 and Fibroblast growth factor-2; FGF-2), indicating the up-regulation of Transforming growth factor-ß (TGF-ß) receptor signaling. Consistent with this, treatment with a specific TGF-ß receptor inhibitor ameliorated both the defects in myotube differentiation and fibrosis. CONCLUSION: Taken together, we demonstrate that SMRT functions as a pivotal transcriptional mediator for both ß-oxidation and the prevention for the fibrosis via TGF-ß receptor signaling in the differentiation of C2C12 myoblasts. In contrast to the results from C2C12 cells, SMRT does not appear to play a role in adult skeletal muscle of whole body SMRT KO mice. Thus, SMRT plays a significant role in the differentiation of myoblasts.