Regulation of phosphosignaling pathways involved in transcription of cell cycle target genes by TRH receptor activation in GH1 cells.

Thyrotropin-releasing hormone (TRH) is known to activate several cellular signaling pathway, but the activation of the TRH receptor (TRH-R) has not been reported to regulate gene transcription. The aim of this study was to identify phosphosignaling pathways and phosphoprotein complexes associated with gene transcription in GH1 pituitary cells treated with TRH or its analog, taltirelin (TAL), using label-free bottom-up mass spectrometry-based proteomics. Our detailed analysis provided insight into the mechanism through which TRH-R activation may regulate the transcription of genes related to the cell cycle and proliferation. It involves control of the signaling pathways for ß-catenin/Tcf, Notch/RBPJ, p53/p21/Rbl2/E2F, Myc, and YY1/Rb1/E2F through phosphorylation and dephosphorylation of their key components. In many instances, the phosphorylation patterns of differentially phosphorylated phosphoproteins in TRH- or TAL-treated cells were identical or displayed a similar trend in phosphorylation. However, some phosphoproteins, especially components of the Wnt/ß-catenin/Tcf and YY1/Rb1/E2F pathways, exhibited different phosphorylation patterns in TRH- and TAL-treated cells. This supports the notion that TRH and TAL may act, at least in part, as biased agonists. Additionally, the deficiency of ß-arrestin2 resulted in a reduced number of alterations in phosphorylation, highlighting the critical role of ß-arrestin2 in the signal transduction from TRH-R in the plasma membrane to transcription factors in the nucleus.

Sex-dependent and -independent regulation of thyrotropin-releasing hormone expression in the hypothalamic dorsomedial nucleus by negative energy balance, exercise, and chronic stress.

The dorsomedial nucleus of the hypothalamus (DMH) is part of the brain circuits that modulate organism responses to the circadian cycle, energy balance, and psychological stress. A large group of thyrotropin-releasing hormone (Trh) neurons is localized in the DMH; they comprise about one third of the DMH neurons that project to the lateral hypothalamus area (LH). We tested their response to various paradigms. In male Wistar rats, food restriction during adulthood, or chronic variable stress (CVS) during adolescence down-regulated adult DMH Trh mRNA levels compared to those in sedentary animals fed ad libitum; two weeks of voluntary wheel running during adulthood enhanced DMH Trh mRNA levels compared to pair-fed rats. Except for their magnitude, female responses to exercise were like those in male rats; in contrast, in female rats CVS did not change DMH Trh mRNA levels. A very strong negative correlation between DMH Trh mRNA levels and serum corticosterone concentration in rats of either sex was lost in CVS rats. CVS canceled the response to food restriction, but not that to exercise in either sex. TRH receptor 1 (Trhr) cells were numerous along the rostro-caudal extent of the medial LH. In either sex, fasting during adulthood reduced DMH Trh mRNA levels, and increased LH Trhr mRNA levels, suggesting fasting may inhibit the activity of TRHDMH->LH neurons. Thus, in Wistar rats DMH Trh mRNA levels are regulated by negative energy balance, exercise and chronic variable stress through sex-dependent and -independent pathways.

IGSF1 Deficiency Leads to Reduced TSH Production Independent of Alterations in Thyroid Hormone Action in Male Mice.

Loss of function mutations in IGSF1/Igsf1 cause central hypothyroidism. Igsf1 knockout mice have reduced pituitary thyrotropin-releasing hormone receptor, Trhr, expression, perhaps contributing to the phenotype. Because thyroid hormones negatively regulate Trhr, we hypothesized that IGSF1 might affect thyroid hormone availability in pituitary thyrotropes. Consistent with this idea, IGSF1 coimmunoprecipitated with the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) in transfected cells. This association was impaired with IGSF1 bearing patient-derived mutations. Wild-type IGSF1 did not, however, alter MCT8-mediated thyroid hormone import into heterologous cells. IGSF1 and MCT8 are both expressed in the apical membrane of the choroid plexus. However, MCT8 protein levels and localization in the choroid plexus were unaltered in Igsf1 knockout mice, ruling out a necessary chaperone function for IGSF1. MCT8 expression was low in the pituitary and was similarly unaffected in Igsf1 knockouts. We next assessed whether IGSF1 affects thyroid hormone transport or action, by MCT8 or otherwise, in vivo. To this end, we treated hypothyroid wild-type and Igsf1 knockout mice with exogenous thyroid hormones. T4 and T3 inhibited TSH release and regulated pituitary and forebrain gene expression similarly in both genotypes. Interestingly, pituitary TSH beta subunit (Tshb) expression was consistently reduced in Igsf1 knockouts relative to wild-type regardless of experimental condition, whereas Trhr was more variably affected. Although IGSF1 and MCT8 can interact in heterologous cells, the physiological relevance of their association is not clear. Nevertheless, the results suggest that IGSF1 loss can impair TSH production independently of alterations in TRHR levels or thyroid hormone action.

Congenital isolated central hypothyroidism: Novel mutations and their functional implications.

Congenital hypothyroidism is the most frequent endocrine disorder in newborns, occurring in 1 per 3000-4000 newborns. In the Netherlands, the neonatal screening program is based primarily on heel prick thyroxine (T4). In contrast to thyroid-stimulating hormone-based programs, this approach allows for the detection of both primary and central congenital hypothyroidism. Over the past decade, the identification of families with isolated congenital central hypothyroidism enabled the identification of novel genetic causes of this condition, in addition to mutations in the TSHß-subunit gene and thyrotropin-releasing hormone receptor gene reported earlier. In 2012, loss-of-function mutations in the immunoglobulin superfamily, member 1 (IGSF1) gene, were reported as a genetic cause of a syndrome including X-linked congenital central hypothyroidism and adult macroorchidism. IGSF1 encodes a hypothalamic plasma membrane glycoprotein. Mutations in IGSF1 represent the most prevalent genetic cause of isolated central hypothyroidism to date. In 2016, mutations in the transducin ß-like 1X (TBL1X) gene were identified in patients with a combination of mild central hypothyroidism and sensorineural hearing loss. TBL1X is an essential subunit of the NCoR/SMRT corepressor complex and expressed in many tissues including the human hypothalamus and pituitary. In 2018, mutations in the insulin receptor substrate 4 (IRS4) gene were reported in cases of familial isolated central hypothyroidism. IRS4 encodes a hypothalamic protein that is part of the insulin and leptin signaling cascade. These recent developments will broaden our understanding of the role of the hypothalamus in hypothalamus-pituitary-thyroid axis regulation and will help to improve diagnosis and treatment of isolated central hypothyroidism.

Novel insights into di­(2­ethylhexyl)phthalate activation: Implications for the hypothalamus­pituitary­thyroid axis.

Di (2­ethylhexyl) phthalate (DEHP), an environmental pollutant, is widely used as a plasticizer and causes serious pollution in the ecological environment. As previously reported, exposure to DEHP may cause thyroid dysfunction of the hypothalamic­pituitary­thyroid (HPT) axis. However, the underlying role of DEHP remains to be elucidated. The present study performed intragastrical administration of DEHP (150, 300 and 600 mg/kg) once a day for 90 consecutive days. DEHP­stimulated oxidative stress increased the thyroid follicular cavity diameter and caused thyrocyte oedema. Furthermore, DEHP exposure altered mRNA and protein levels. Thus, DEHP may perturb TH homeostasis by affecting biosynthesis, biotransformation, bio­transportation, receptor levels and metabolism through disruption of the HPT axis and activation of the thyroid­stimulating hormone (TSH)/TSH receptor signaling pathway. These results identified the formerly unappreciated endocrine­disrupting activities of phthalates and the molecular mechanisms of DEHP­induced thyrotoxicity.

Sexually dimorphic dynamics of thyroid axis activity during fasting in rats.

Starvation induces tertiary hypothyroidism in adult rodents. Response of the hypothalamus-pituitary-thyroid (HPT) axis to starvation is stronger in adult males than in females. To improve the description of this sexual dimorphism, we analyzed the dynamics of HPT axis response to fasting at multiple levels. In adult rats of the same cohort, 24 and 48 h of starvation inhibited paraventricular nucleus Trh expression and serum concentrations of TSH and T4 earlier in males than in females, with lower intensity in females than in males. In adult females fasted for 36-72 h, serum TSH concentration decreased after 36 h, when the activity of thyrotropin-releasing hormone (TRH)-degrading ectoenzyme was increased in the median eminence. The kinetics of these events were distinct from those previously observed in male rats. We suggest that the sex difference in TSH secretion kinetics is driven not only at the level of paraventricular nucleus TRH neurons, but also by differences in post-secretory catabolism of TRH, with enhancement of TRH-degrading activity more sustained in male than female animals.

Characterization of a novel thyrotropin-releasing hormone receptor, TRHR3, in chickens.

The physiological roles of thyrotropin-releasing hormone (TRH) are proposed to be mediated by TRH receptors (TRHR), which have been divided into 3 subtypes, namely, TRHR1, TRHR2, and TRHR3, in vertebrates. Although 2 TRH receptors (TRHR1 and TRHR3) have been predicted to exist in birds, it remains unclear whether TRHR3 is a functional TRH receptor similar to TRHR1. Here, we reported the functionality and tissue expression of TRHR3 in chickens. The cloned chicken TRHR3 (cTRHR3) encodes a receptor of 387 amino acids, which shares high-amino-acid identities (63-80%) to TRHR3 of parrots, lizards, Xenopus tropicalis, and tilapia and comparatively lower sequence identities to chicken TRHR1 or mouse TRHR2. Using cell-based luciferase reporter assays and Western blot, we demonstrated that similar to chicken TRHR1 (cTRHR1), cTRHR3 expressed in HEK 293 cells can be potently activated by TRH and that its activation stimulates multiple signaling pathways, indicating both TRH receptors are functional. Quantitative real-time PCR revealed that cTRHR1 and cTRHR3 are widely, but differentially, expressed in chicken tissues, and their expression is likely controlled by promoters located upstream of exon 1, which display strong promoter activities in cultured DF-1 cells. cTRHR1 is highly expressed in the anterior pituitary and testes, while cTRHR3 is highly expressed in the muscle, testes, fat, pituitary, spinal cord, and many brain regions (including hypothalamus). These findings indicate that TRH actions are likely mediated by 2 TRH receptors in chickens. In conclusion, our data provide the first piece of evidence that both cTRHR3 and cTRHR1 are functional TRH receptors, which helps to elucidate the physiological roles of TRH in birds.

Genetics of Congenital Isolated TSH Deficiency: Mutation Screening of the Known Causative Genes and a Literature Review.

CONTEXT: Congenital isolated TSH deficiency (i-TSHD) is a rare form of congenital hypothyroidism. Five genes (IGSF1, IRS4, TBL1X, TRHR, and TSHB) responsible for the disease have been identified, although their relative frequencies and hypothalamic/pituitary unit phenotypes have remained to be clarified. OBJECTIVES: To define the relative frequencies and hypothalamic/pituitary unit phenotypes of congenital i-TSHD resulting from single gene mutations. PATIENTS AND METHODS: Thirteen Japanese patients (11 boys and 2 girls) with congenital i-TSHD were enrolled. IGSF1, IRS4, TBL1X, TRHR, and TSHB were sequenced. For a TBL1X mutation (p.Asn382del), its pathogenicity was verified in vitro. For a literature review, published clinical data derived from 74 patients with congenital i-TSHD resulting from single-gene mutations were retrieved and analyzed. RESULTS: Genetic screening of the 13 study subjects revealed six mutation-carrying patients (46%), including five hemizygous IGSF1 mutation carriers and one hemizygous TBL1X mutation carrier. Among the six mutation carriers, one had intellectual disability and the other one had obesity, but the remaining four did not show nonendocrine phenotypes. Loss of function of the TBL1X mutation (p.Asn382del) was confirmed in vitro. The literature review demonstrated etiology-specific relationship between serum prolactin (PRL) levels and TRH-stimulated TSH levels with some degree of overlap. CONCLUSIONS: The mutation screening study covering the five causative genes of congenital i-TSHD was performed, showing that the IGSF1 defect was the leading genetic cause of the disease. Assessing relationships between serum PRL levels and TRH-stimulated TSH levels would contribute to predict the etiologies of congenital i-TSHD.

Possible involvement of thyrotropin-releasing hormone receptor 3 in the release of prolactin in the metamorphosing bullfrog larvae.

In amphibians, thyrotropin (TSH), corticotropin (ACTH) and prolactin (PRL) are regarded as the major pituitary hormones involved in metamorphosis, their releasing factors being corticotropin-releasing factor (CRF), arginine vasotocin (AVT), and thyrotropin-releasing hormone (TRH), respectively. It is also known that thyrotropes and corticotropes are equipped with CRF type-2 receptor and AVT V1b receptor, respectively. As for PRL cells, information about the type of receptor for TRH (TRHR) through which the action of TRH is mediated to induce the release of PRL is lacking. In order to fill this gap, an attempt was made to characterize the TRHR subtype existing in the PRL cells of the anterior pituitary gland of the bullfrog, Rana catesbeiana. We cloned cDNAs for three types of bullfrog TRHRs, namely TRHR1, TRHR2 and TRHR3, and confirmed that all of them are functional receptors for TRH by means of reporter gene assay. Analyses with semi-quantitative reverse transcription-PCR and in situ hybridization revealed that TRHR3 mRNA is expressed in the anterior lobe and that the signals reside mostly in the PRL cells. It was also noted that the expression levels of TRHR3 mRNA in the anterior pituitary as well as in the PRL cells of metamorphosing tadpoles elevate as metamorphosis progresses. Since the pattern of changes in TRHR3 mRNA levels in the larval pituitary is almost similar to that previously observed in the pituitary PRL mRNA and plasma PRL levels, we provide a view that TRHR3 mediates the action of TRH on the PRL cells to induce the release of PRL that is prerequisite for growth and metamorphosis in amphibians.

P38/TRHr-Dependent Regulation of TPO in Thyroid Cells Contributes to the Hypothyroidism of Triclosan-Treated Rats.

BACKGROUND/AIMS: Triclosan, as an antimicrobial agent and a potential endocrine disruptor, has been used extensively in diverse products, resulting in widespread human exposure. In recent years, studies suggest that triclosan could disturb thyroid functions and decline thyroid hormones (THs). METHODS: To verify our hypothesis that the MAPK pathway may function significantly in triclosan-induced hypothyroidism, Sprague-Dawley rats were gavaged with triclosan for 31 consecutive days; Nthy-ori 3-1 cells were treated with triclosan in the presence/absence of NAC, inhibitors (SB203580 and SB202474), or TRHr siRNA. Tissues and/or cells were analyzed by several techniques including transmission electron microscopy, confocal laser scanning microscopy, gene silencing, western blot, and real-time PCR. RESULTS: Triclosan led to histopathologic changes in the thyroid and decreases in triiodothyronine (T3) and thyroxine (T4). Triclosan stimulated ROS production and oxidative stress occurrence, thereby activating the p38 pathway in vivo and in vitro. Thyrotropin releasing hormone receptor (TRHr) was induced when the p38 pathway was activated, and was suppressed when that pathway was inhibited. Moreover, thyroid peroxidase (TPO) was restrained and modulated by the p38/TRHr pathway after triclosan treatment. Furthermore, deiodinase 3 (D3) and hepatic enzymes (Ugt2b1, CYP1a1, CYP1a2, CYP2b1, CYP3a1, and Sult1e1) were also induced by triclosan. CONCLUSION: Taken together, p38/TRHr-dependent regulation of TPO in thyroid cells contributes to the hypothyroidism of triclosan-treated rats.

Neonatal exposure to bisphenol A alters the hypothalamic-pituitary-thyroid axis in female rats.

Bisphenol A (BPA) is a component of polycarbonate plastics, epoxy resins and polystyrene found in many common products. Several reports revealed potent in vivo and in vitro effects. In this study we analyzed the effects of the exposure to BPA in the hypothalamic-pituitary-thyroid axis in female rats, both in vivo and in vitro. Female Sprague-Dawley rats were injected sc from postnatal day 1 (PND1) to PND10 with BPA: 500 µg 50 µl-1 oil (B500), or 50 µg 50 µl-1 (B50), or 5 µg 50 µl-1 (B5). Controls were injected with 50 µl vehicle during the same period. Neonatal exposure to BPA did not modify TSH levels in PND13 females, but it increased them in adults in estrus. Serum T4 was lower in B5 and B500 with regards to Control, whereas no difference was seen in T3. No significant differences were observed in TRH, TSHß and TRH receptor expression between groups. TSH release from PPC obtained from adults in estrus was also higher in B50 with regard to Control. In vitro 24 h pre-treatment with BPA or E2 increased basal TSH as well as prolactin release. On the other hand, both BPA and E2 lowered the response to TRH. The results presented here show that the neonatal exposure to BPA alters the hypothalamic pituitary-thyroid axis in adult rats in estrus, possibly with effects on the pituitary and thyroid. They also show that BPA alters TSH release from rat PPC through direct actions on the pituitary.

High-fat, simple-carbohydrate diet intake induces hypothalamic-pituitary-thyroid axis dysregulation in C57BL/6J male mice.

Given the association between subclinical hypothyroidism and metabolic syndrome, we wanted to explore if high-fat, simple-carbohydrate (HFSC) diet affects hypothalamus-pituitary-thyroid axis. One-month-old male C57BL/6J mice were fed with control (C) and HFSC (T) feed (n = 18 each), respectively, for 5 months. There was a significant increase in triiodothyronine in the T group (13.5%) compared with the age-matched C group by the fifth month. Thyroid-stimulating hormone was significantly higher (1 month: 1.9-fold; 3 months: 2.66-fold; 5 months: 3.5-fold) from the first to fifth months in the T group compared with age-matched C group. Thyrotropin-releasing hormone (TRH) gene expression showed significant decrease (1 month: 83.2%; 5 months: 40.7%) in the T group compared with the age-matched C group. TRHR1 showed significant decrease in the T group compared with the age-matched C group throughout the study (1 month: 82.8%; 3 months: 45.7%; 5 months: 75.2%). However, TRHR2 showed dynamic change during the study. Initially there was significant (1 month: 0.104-fold) downregulation, followed by significant upregulation (3 months: 3.6-fold) and downregulation (0.73-fold) by the fifth month in the T group compared with the age-matched C group. There was marked depletion of functional follicular cells and colloid substance in the thyroid glands of the T group by the fifth month compared with the C group. Leptin receptors ObRa (1 month: 48.25%; 5 months: 88%) and ObRb (1 month: 46.9%; 5 months: 63.3%) were significantly downregulated in the T group compared with the age-matched C group in the first and fifth months of feeding the respective diets. The expression of p-STAT3, a transcription factor known to have a role in energy balance, intermediate metabolism, and leptin signalling was seen to decrease significantly (6.25-fold) in the hypothalamus of the T group compared with the age-matched C group. In conclusion, HFSC feed disrupts the hypothalamus-pituitary-thyroid axis in male C57BL/6J mice.

TRH receptor mobility in the plasma membrane is strongly affected by agonist binding and by interaction with some cognate signaling proteins.

OBJECTIVES: Extensive research has been dedicated to elucidating the mechanisms of signal transduction through different G protein-coupled receptors (GPCRs). However, relatively little is known about the regulation of receptor movement within the cell membrane upon ligand binding. In this study we focused our attention on the thyrotropin-releasing hormone (TRH) receptor that typically couples to Gq/11 proteins. METHODS: We monitored receptor diffusion in the plasma membrane of HEK293 cells stably expressing yellow fluorescent protein (YFP)-tagged TRH receptor (TRHR-YFP) by fluorescence recovery after photobleaching (FRAP). RESULTS: FRAP analysis indicated that the lateral movement of the TRH receptor was markedly reduced upon TRH binding as the value of its diffusion coefficient fell down by 55%. This effect was prevented by the addition of the TRH receptor antagonist midazolam. We also found that siRNA-mediated knockdown of Gq/11α, Gß, ß-arrestin2 and phospholipase Cß1, but not of Giα1, ß-arrestin1 or G protein-coupled receptor kinase 2, resulted in a significant decrease in the rate of TRHR-YFP diffusion, indicating the involvement of the former proteins in the regulation of TRH receptor behavior. The observed partial reduction of the TRHR-YFP mobile fraction caused by down-regulation of Giα1 and ß-arrestin1 suggests that these proteins may also play distinct roles in THR receptor-mediated signaling. CONCLUSION: These results demonstrate for the first time that not only agonist binding but also abundance of some signaling proteins may strongly affect TRH receptor dynamics in the plasma membrane.

Visual Neurons in the Superior Colliculus Innervated by Islet2+ or Islet2- Retinal Ganglion Cells Display Distinct Tuning Properties.

Throughout the visual system, different subtypes of neurons are tuned to distinct aspects of the visual scene, establishing parallel circuits. Defining the mechanisms by which such tuning arises has been a long-standing challenge for neuroscience. To investigate this, we have focused on the retina's projection to the superior colliculus (SC), where multiple visual neuron subtypes have been described. The SC receives inputs from a variety of retinal ganglion cell (RGC) subtypes; however, which RGCs drive the tuning of different SC neurons remains unclear. Here, we pursued a genetic approach that allowed us to determine the tuning properties of neurons innervated by molecularly defined subpopulations of RGCs. In homozygous Islet2-EphA3 knock-in (Isl2EA3/EA3) mice, Isl2+ and Isl2- RGCs project to non-overlapping sub-regions of the SC. Based on molecular and anatomic data, we show that significantly more Isl2- RGCs are direction-selective (DS) in comparison with Isl2+ RGCs. Targeted recordings of visual responses from each SC sub-region in Isl2EA3/EA3 mice revealed that Isl2- RGC-innervated neurons were significantly more DS than those innervated by Isl2+ RGCs. Axis-selective (AS) neurons were found in both sub-regions, though AS neurons innervated by Isl2+ RGCs were more tightly tuned. Despite this segregation, DS and AS neurons innervated by Isl2+ or Isl2- RGCs did not differ in their spatial summation or spatial frequency (SF) tuning. Further, we did not observe alterations in receptive field (RF) size or structure of SC neurons innervated by Isl2+ or Isl2- RGCs. Together, these data show that innervation by Isl2+ and Isl2- RGCs results in distinct tuning in the SC and set the stage for future studies investigating the mechanisms by which these circuits are built.

Tanycytes control the hormonal output of the hypothalamic-pituitary-thyroid axis.

The hypothalamic-pituitary-thyroid (HPT) axis maintains circulating thyroid hormone levels in a narrow physiological range. As axons containing thyrotropin-releasing hormone (TRH) terminate on hypothalamic tanycytes, these specialized glial cells have been suggested to influence the activity of the HPT axis, but their exact role remained enigmatic. Here, we demonstrate that stimulation of the TRH receptor 1 increases intracellular calcium in tanycytes of the median eminence via Gαq/11 proteins. Activation of Gαq/11 pathways increases the size of tanycyte endfeet that shield pituitary vessels and induces the activity of the TRH-degrading ectoenzyme. Both mechanisms may limit the TRH release to the pituitary. Indeed, blocking TRH signaling in tanycytes by deleting Gαq/11 proteins in vivo enhances the response of the HPT axis to the chemogenetic activation of TRH neurons. In conclusion, we identify new TRH- and Gαq/11-dependent mechanisms in the median eminence by which tanycytes control the activity of the HPT axis.The hypothalamic-pituitary-thyroid (HPT) axis regulates a wide range of physiological processes. Here the authors show that hypothalamic tanycytes play a role in the homeostatic regulation of the HPT axis; activation of TRH signaling in tanycytes elevates their intracellular Ca2+ via Gαq/11 pathway, ultimately resulting in reduced TRH release into the pituitary vessels.

Taltirelin alleviates fatigue-like behavior in mouse models of cancer-related fatigue.

Fatigue affects most cancer patients and has numerous potential causes, including cancer itself and cancer treatment. Cancer-related fatigue (CRF) is not relieved by rest, can decrease quality of life, and has no FDA-approved therapy. Thyrotropin-releasing hormone (TRH) has been proposed as a potential novel treatment for CRF, but its efficacy against CRF remains largely untested. Thus, we tested the TRH analog, taltirelin (TAL), in mouse models of CRF. To model fatigue, we used a mouse model of chemotherapy, a mouse model of radiation therapy, and mice bearing colon 26 carcinoma tumors. We used the treadmill fatigue test to assess fatigue-like behavior after treatment with TAL. Additionally, we used wild-type and TRH receptor knockout mice to determine which TRH receptor was necessary for the actions of TAL. Tumor-bearing mice displayed muscle wasting and all models caused fatigue-like behavior, with mice running a shorter distance in the treadmill fatigue test than controls. TAL reversed fatigue-like behavior in all three models and the mouse TRH1 receptor was necessary for the effects of TAL. These data suggest that TAL may be useful in alleviating fatigue in all cancer patients and provide further support for evaluating TAL as a potential therapy for CRF in humans.

Central Hypothyroidism Due to a TRHR Mutation Causing Impaired Ligand Affinity and Transactivation of Gq.

Context: Central congenital hypothyroidism (CCH) is an underdiagnosed disorder characterized by deficient production and bioactivity of thyroid-stimulating hormone (TSH) leading to low thyroid hormone synthesis. Thyrotropin-releasing hormone (TRH) receptor (TRHR) defects are rare recessive disorders usually associated with incidentally identified CCH and short stature in childhood. Objectives: Clinical and genetic characterization of a consanguineous family of Roma origin with central hypothyroidism and identification of underlying molecular mechanisms. Design: All family members were phenotyped with thyroid hormone profiles, pituitary magnetic resonance imaging, TRH tests, and dynamic tests for other pituitary hormones. Candidate TRH, TRHR, TSHB, and IGSF1 genes were screened for mutations. A mutant TRHR was characterized in vitro and by molecular modeling. Results: A homozygous missense mutation in TRHR (c.392T > C; p.I131T) was identified in an 8-year-old boy with moderate hypothyroidism (TSH: 2.61 mIU/L, Normal: 0.27 to 4.2; free thyroxine: 9.52 pmol/L, Normal: 10.9 to 25.7) who was overweight (body mass index: 20.4 kg/m2, p91) but had normal stature (122 cm; -0.58 standard deviation). His mother, two brothers, and grandmother were heterozygous for the mutation with isolated hyperthyrotropinemia (TSH: 4.3 to 8 mIU/L). The I131T mutation, in TRHR intracellular loop 2, decreases TRH affinity and increases the half-maximal effective concentration for signaling. Modeling of TRHR-Gq complexes predicts that the mutation disrupts the interaction between receptor and a hydrophobic pocket formed by Gq. Conclusions: A unique missense TRHR defect identified in a consanguineous family is associated with central hypothyroidism in homozygotes and hyperthyrotropinemia in heterozygotes, suggesting compensatory elevation of TSH with reduced biopotency. The I131T mutation decreases TRH binding and TRHR-Gq coupling and signaling.

The syndrome of central hypothyroidism and macroorchidism: IGSF1 controls TRHR and FSHB expression by differential modulation of pituitary TGFß and Activin pathways.

IGSF1 (Immunoglobulin Superfamily 1) gene defects cause central hypothyroidism and macroorchidism. However, the pathogenic mechanisms of the disease remain unclear. Based on a patient with a full deletion of IGSF1 clinically followed from neonate to adulthood, we investigated a common pituitary origin for hypothyroidism and macroorchidism, and the role of IGSF1 as regulator of pituitary hormone secretion. The patient showed congenital central hypothyroidism with reduced TSH biopotency, over-secretion of FSH at neonatal minipuberty and macroorchidism from 3 years of age. His markedly elevated inhibin B was unable to inhibit FSH secretion, indicating a status of pituitary inhibin B resistance. We show here that IGSF1 is expressed both in thyrotropes and gonadotropes of the pituitary and in Leydig and germ cells in the testes, but at very low levels in Sertoli cells. Furthermore, IGSF1 stimulates transcription of the thyrotropin-releasing hormone receptor (TRHR) by negative modulation of the TGFß1-Smad signaling pathway, and enhances the synthesis and biopotency of TSH, the hormone secreted by thyrotropes. By contrast, IGSF1 strongly down-regulates the activin-Smad pathway, leading to reduced expression of FSHB, the hormone secreted by gonadotropes. In conclusion, two relevant molecular mechanisms linked to central hypothyroidism and macroorchidism in IGSF1 deficiency are identified, revealing IGSF1 as an important regulator of TGFß/Activin pathways in the pituitary.

TRH Action Is Impaired in Pituitaries of Male IGSF1-Deficient Mice.

Loss-of-function mutations in the X-linked immunoglobulin superfamily, member 1 (IGSF1) gene cause central hypothyroidism. IGSF1 is a transmembrane glycoprotein of unknown function expressed in thyrotropin (TSH)-producing thyrotrope cells of the anterior pituitary gland. The protein is cotranslationally cleaved, with only its C-terminal domain (CTD) being trafficked to the plasma membrane. Most intragenic IGSF1 mutations in humans map to the CTD. In this study, we used CRISPR-Cas9 to introduce a loss-of-function mutation into the IGSF1-CTD in mice. The modified allele encodes a truncated protein that fails to traffic to the plasma membrane. Under standard laboratory conditions, Igsf1-deficient males exhibit normal serum TSH levels as well as normal numbers of TSH-expressing thyrotropes. However, pituitary expression of the TSH subunit genes and TSH protein content are reduced, as is expression of the receptor for thyrotropin-releasing hormone (TRH). When challenged with exogenous TRH, Igsf1-deficient males release TSH, but to a significantly lesser extent than do their wild-type littermates. The mice show similarly attenuated TSH secretion when rendered profoundly hypothyroid with a low iodine diet supplemented with propylthiouracil. Collectively, these results indicate that impairments in pituitary TRH receptor expression and/or downstream signaling underlie central hypothyroidism in IGSF1 deficiency syndrome.

Hypothalamo­hypophysial system in rats with autotransplantation of the adrenal cortex.

Patients with bilateral pheochromocytoma often require an adrenalectomy. Autotransplantation of the adrenal cortex is an alternative therapy that could potentially be performed instead of receiving glucocorticoid replacement following adrenalectomy. Adrenal cortex autotransplantation aims to avoid the side effects of long­term steroid treatment and adrenal insufficiency. Although the function of the hypothalamo­hypophysial system is critical for patients who have undergone adrenal cortex autotransplantation, the details of that system, with the exception of adrenocorticotropic hormone in the subjects with adrenal autotransplantation, have been overlooked for a long time. To clarify the precise effect of adrenal autotransplantation on the pituitary gland and hypothalamus, the current study examined the gene expression of hormones produced from the hypothalamus and pituitary gland. Bilateral adrenalectomy and adrenal autotransplantation were performed in 8 to 9­week­old male rats. The hypothalamus and pituitary tissues were collected at 4 weeks after surgery. Transcriptional regulation of hypothalamic and pituitary hormones was subsequently examined by reverse transcription­quantitative polymerase chain reaction. Proopiomelanocortin, glycoprotein hormone α polypeptide, and thyroid stimulating hormone ß were significantly elevated in the pituitary gland of autotransplanted rats when compared with sham­operated rats. In addition, there were significant differences in the levels of corticotropin releasing hormone receptor 1 (Crhr1), Crhr2, nuclear receptor subfamily 3 group C member 1 and thyrotropin releasing hormone receptor between the sham­operated rats and autotransplanted rats in the pituitary gland. In the hypothalamus, corticotropin releasing hormone and urocortin 2 mRNA was significantly upregulated in autotransplanted rats compared with sham­operated rats. The authors identified significant alterations in the function of not only the hypothalamus­pituitary­adrenal axis, but also the adenohypophysis thyrotropes in autotransplanted rats. In the future, it will be important to examine other tissues affected by glucocorticoids following adrenal cortex autotransplantation.