Search

Thyroid transcription factors (TTFs) - NKX2-1, FOXE1, PAX8 and HHEX - regulate multiple genes involved in thyroid development in mice but little is known about TTF regulation of thyroid-specific genes - thyroglobulin (TG), thyroid peroxidase (TPO), deiodinase type 2 (DIO2), sodium/iodide symporter (NIS) and TSH receptor (TSHR) - in adult, human thyrocytes. Thyrotropin (thyroid-stimulating hormone, TSH) regulation of thyroid-specific gene expression in primary cultures of human thyrocytes is biphasic yielding an inverted U-shaped dose-response curve (IUDRC) with upregulation at low doses and decreases at high doses. Herein we show that NKX2-1, FOXE1 and PAX8 are required for TSH-induced upregulation of the mRNA levels of TG, TPO, DIO2, NIS, and TSHR whereas HHEX has little effect on the levels of these thyroid-specific gene mRNAs. We show that TSH-induced upregulation is mediated by changes in their transcription and not by changes in the degradation of their mRNAs. In contrast to the IUDRC of thyroid-specific genes, TSH effects on the levels of the mRNAs for NKX2-1, FOXE1 and PAX8 exhibit monophasic decreases at high doses of TSH whereas TSH regulation of HHEX mRNA levels exhibits an IUDRC that overlaps the IUDRC of thyroid-specific genes. In contrast to findings during mouse development, TTFs do not have major effects on the levels of other TTF mRNAs in adult, human thyrocytes. Thus, we found similarities and important differences in the regulation of thyroid-specific genes in mouse development and TSH regulation of these genes in adult, human thyrocytes. • TSH upregulation of thyroid-specific genes is dependent on NKX2-1, FOXE1 and PAX8, but not HHEX. • TSH regulation of thyroid-specific genes is mediated by changes in their transcription and not by the degradation of mRNAs. • TSH causes monophasic inhibition of NKX2-1, FOXE1 and PAX8 mRNA but an IUDRC for HHEX mRNA. • TTFs do not have major effects on the levels of other TTF mRNAs in adult, human thyrocytes. [ABSTRACT FROM AUTHOR]

Context: Thyroid-stimulating hormone (or thyrotropin) receptor (TSHR) could be a selective target for small molecule ligands to treat thyroid cancer (TC)., Objective: We report a novel, orally efficacious ligand for TSHR that exhibits proliferation inhibitory activity against human TC in vitro and in vivo, and inhibition of metastasis in vivo., Methods: A35 (NCATS-SM4420; NCGC00241808) was selected from a sublibrary of >200 TSHR ligands. Cell proliferation assays including BrdU incorporation and WST-1, along with molecular docking studies were done. In vivo activity of A35 was assessed in TC cell-derived xenograft (CDX) models with immunocompromised (NSG) mice. Formalin-fixed, paraffin-embedded sections of tumor and lung tissues were observed for the extent of cell death and metastasis., Results: A35 was shown to stimulate cAMP production in some cell types by activating TSHR but not in TC cells, MDA-T32, and MDA-T85. A35 inhibited proliferation of MDA-T32 and MDA-T85 in vitro and in vivo, and pulmonary metastasis of MDA-T85F1 in mice. In vitro, A35 inhibition of proliferation was reduced by a selective TSHR antagonist. Inhibition of CDX tumor growth without decreases in mouse weights and liver function showed A35 to be efficacious without apparent toxicity. Lastly, A35 reduced levels of Ki67 in the tumors and metastatic markers in lung tissues., Conclusion: We conclude that A35 is a TSHR-selective inhibitor of TC cell proliferation and metastasis, and suggest that A35 may be a promising lead drug candidate for the treatment of differentiated TC in humans., (Published by Oxford University Press on behalf of the Endocrine Society 2024.)

Background: Astrocytes respond to injury and disease through a process known as reactive astrogliosis, of which inflammatory signaling is one subset. This inflammatory response is heterogeneous with respect to the inductive stimuli and the afflicted central nervous system region. This is of plausible importance in e.g. traumatic axonal injury (TAI), where lesions in the brainstem carries a particularly poor prognosis. In fact, astrogliotic forebrain astrocytes were recently suggested to cause neuronal death following axotomy. We therefore sought to assess if ventral brainstem- or rostroventral spinal astrocytes exert similar effects on motor neurons in vitro., Methods: We derived brainstem/rostroventral spinal astrocyte-like cells (ES-astrocytes) and motor neurons using directed differentiation of mouse embryonic stem cells (ES). We activated the ES-astrocytes using the neurotoxicity-eliciting cytokines interleukin- (IL-) 1α and tumor necrosis factor-(TNF-)α and clinically relevant inflammatory mediators. In co-cultures with reactive ES-astrocytes and motor neurons, we assessed neurotoxic ES-astrocyte activity, similarly to what has previously been shown for other central nervous system (CNS) regions., Results: We confirmed the brainstem/rostroventral ES-astrocyte identity using RNA-sequencing, immunocytochemistry, and by comparison with primary subventricular zone-astrocytes. Following cytokine stimulation, the c-Jun N-terminal kinase pathway down-stream product phosphorylated c-Jun was increased, thus demonstrating ES-astrocyte reactivity. These reactive ES-astrocytes conferred a contact-dependent neurotoxic effect upon co-culture with motor neurons. When exposed to IL-1β and IL-6, two neuroinflammatory cytokines found in the cerebrospinal fluid and serum proteome following human severe traumatic brain injury (TBI), ES-astrocytes exerted similar effects on motor neurons. Activation of ES-astrocytes by these cytokines was associated with pathways relating to endoplasmic reticulum stress and altered regulation of MYC., Conclusions: Ventral brainstem and rostroventral spinal cord astrocytes differentiated from mouse ES can exert neurotoxic effects in vitro. This highlights how neuroinflammation following CNS lesions can exert region- and cell-specific effects. Our in vitro model system, which uniquely portrays astrocytes and neurons from one niche, allows for a detailed and translationally relevant model system for future studies on how to improve neuronal survival in particularly vulnerable CNS regions following e.g. TAI., (© 2023. The Author(s).)

Regulation of thyroid cells by thyrotropin (TSH) and epidermal growth factor (EGF) has been known but different effects of these regulators on proliferation and differentiation have been reported. We studied these responses in primary cultures of human thyroid cells to determine whether TSH receptor (TSHR) signaling may involve EGF receptor (EGFR) transactivation. We confirm that EGF stimulates proliferation and de-differentiation whereas TSH causes differentiation in the absence of other growth factors. We show that TSH/TSHR transactivates EGFR and characterize it as follows: (1) TSH-induced upregulation of thyroid-specific genes is inhibited by 2 inhibitors of EGFR kinase activity, AG1478 and erlotinib; (2) the mechanism of transactivation is independent of an extracellular EGFR ligand by showing that 2 antibodies, cetuximab and panitumumab, that completely inhibited binding of EGFR ligands to EGFR had no effect on transactivation, and by demonstrating that no EGF was detected in media conditioned by thyrocytes incubated with TSH; (3) TSH/TSHR transactivation of EGFR is different than EGFR activation by EGF by showing that EGF led to rapid phosphorylation of EGFR whereas transactivation occurred in the absence of receptor phosphorylation; (4) EGF caused downregulation of EGFR whereas transactivation had no effect on EGFR level; (5) EGF and TSH stimulation converged on the protein kinase B (AKT) pathway, because TSH, like EGF, stimulated phosphorylation of AKT that was inhibited by EGFR inhibitors; and (6) TSH-induced upregulation of thyroid genes was inhibited by the AKT inhibitor MK2206. Thus, TSH/TSHR causes EGFR transactivation that is independent of extracellular EGFR ligand and in part mediates TSH regulation of thyroid hormone biosynthetic genes., (Published by Oxford University Press on behalf of the Endocrine Society 2022.)

Proximity ligation assay (PLA) is a methodology that permits detection of protein-protein closeness, that is, proteins that are within 40 nanometers of each other, in cells or tissues at endogenous protein levels or after exogenous overexpression. It detects the protein(s) with high sensitivity and specificity because it employs a DNA hybridization step followed by DNA amplification. PLA has been used successfully with many types of proteins. In this methods paper, we will describe the workings of PLA and provide examples of its use to study TSH/IGF-1 receptor crosstalk in Graves' orbital fibroblasts (GOFs) and TSH receptor homodimerization in primary cultures of human thyrocytes., Competing Interests: MCG is a consultant with Crosstalk Therapeutics LTD. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2022 Krieger, Boutin, Neumann and Gershengorn.)

Context: We previously presented evidence that TSH receptor (TSHR)-stimulating autoantibodies (TSAbs) bind to and activate TSHRs but do not bind to IGF1 receptors (IGF1Rs). Nevertheless, we showed that IGF1Rs were involved in thyroid eye disease (TED) pathogenesis because TSAbs activated crosstalk between TSHR and IGF1R. Teprotumumab, originally generated to inhibit IGF1 binding to IGF1R, was recently approved for the treatment of TED (Tepezza)., Objective: To investigate the role of TSHR/IGF1R crosstalk in teprotumumab treatment of TED., Design: We used orbital fibroblasts from patients with TED (TEDOFs) and measured stimulated hyaluronan (HA) secretion as a measure of orbital fibroblast activation by TED immunoglobulins (TED-Igs) and monoclonal TSAb M22. We previously showed that M22, which does not bind to IGF1R, stimulated HA in a biphasic dose-response with the higher potency phase dependent on TSHR/IGF1R crosstalk and the lower potency phase independent of IGF1R. Stimulation by TED-Igs and M22 was measured in the absence or presence of teprotumumab biosimilar (Tepro) or K1-70, an antibody that inhibits TSHR., Results: We show: (1) Tepro dose-dependently inhibits stimulation by TED-Igs; (2) Tepro does not bind to TSHRs; (3) Tepro inhibits IGF1R-dependent M22-induced HA production, which is mediated by TSHR/IGF1R crosstalk, but not IGF1R-independent M22 stimulation; and (4) β-arrestin 1 knockdown, which blocks TSHR/IGF1R crosstalk and prevents Tepro inhibition of HA production by M22 and by a pool of TED-Igs., Conclusion: We conclude that Tepro inhibits HA production by TEDOFs by inhibiting TSHR/IGF1R crosstalk and suggest that inhibition of TSHR/IGF1R crosstalk is the mechanism of its action in treating TED., (Published by Oxford University Press on behalf of the Endocrine Society 2021.)

Graves' disease (GD) is an autoimmune disease caused in part by thyroid-stimulating antibodies (TSAbs) that activate the thyroid-stimulating hormone receptor (TSHR). In Graves' hyperthyroidism (GH), TSAbs cause persistent stimulation of thyroid cells leading to continuous thyroid hormone synthesis and secretion. Thyroid eye disease (TED), also called Graves' orbitopathy, is an orbital manifestation of GD. We review the important roles of the TSHR and the insulin-like growth factor 1 receptor (IGF-1R) in the pathogenesis of TED and discuss a model of TSHR/IGF-1R crosstalk that considers two pathways initiated by TSAb activation of TSHR in the eye, an IGF-1R-independent and an IGF-1R-dependent signaling pathway leading to hyaluronan (HA) secretion in orbital fibroblasts. We discuss current and future therapeutic approaches targeting the IGF-1R and TSHR. Teprotumumab, a human monoclonal anti-IGF-1R-blocking antibody, has been approved as an effective treatment in patients with TED. However, as the TSHR seems to be the primary target for TSAbs in patients with GD, future therapeutic interventions directly targeting the TSHR, e.g. blocking antibodies and small molecule antagonists, are being developed and have the advantage to inhibit the IGF-1R-independent as well as the IGF-1R-dependent component of TSAb-induced HA secretion. Antigen-specific immunotherapies using TSHR peptides to reduce serum TSHR antibodies are being developed also. These TSHR-targeted strategies also have the potential to treat both GH and TED with the same drug. We propose that combination therapy targeting TSHR and IGF-1R may be an effective and better tolerated treatment strategy for TED., Competing Interests: Christine Carag Krieger, Susanne Neumann, and Marvin Carl Gershengorn have filed a patent pertaining to drug combinations targeting TSHR and IGF-1R (application No.: KS Ref. 4239-93897-01, E-No. E–284–2008/1)., (Copyright © 2020 by S. Karger AG, Basel.)

Regulating gene expression at the protein level is becoming increasingly important for answering basic questions in neurobiology. Several techniques using destabilizing domains (DD) on transgenes, which can be activated or deactivated by specific drugs, have been developed to achieve this goal. A DD from bacterial dihydrofolate reductase bound and stabilized by trimethoprim (TMP) represents such a tool. To control transgenic protein levels in the brain, the DD-regulating drugs need to have sufficient penetration into the central nervous system (CNS). Yet, very limited information is available on TMP pharmacokinetics in the CNS following systemic injection. Here, we performed a pharmacokinetic study on the penetration of TMP into different CNS compartments in the rat. We used mass spectrometry to measure TMP concentrations in serum, cerebrospinal fluid (CSF) and tissue samples of different CNS regions upon intraperitoneal TMP injection. We show that TMP quickly (within 10 min) penetrates from serum to CSF through the blood-CSF barrier. TMP also shows quick penetration into brain tissue but concentrations were an order of magnitude lower compared to serum or CSF. TMP concentration in spinal cord was lower than in any other analyzed CNS area. Nevertheless, effective levels of TMP to stabilize DDs can be reached in the CNS with half-lives around 2 h. These data show that TMP has good and fast penetration properties into the CNS and is therefore a valuable ligand for precisely controlling protein expression in the CNS in rodents., (Copyright © 2020 Ineichen, Di Palma, Laczko, Liddelow, Neumann, Schwab and Mosberger.)

Cranial irradiation (IR) is commonly used to treat primary brain tumors and metastatic diseases. However, cranial IR-treated patients often develop vascular abnormalities later in life that increase their risk for cerebral ischemia. Studies in rodents have demonstrated that IR impairs maintenance of the neural stem/precursor cell (NSPC) pool and depletes neurogenesis. We and others have previously shown that stroke triggers NSPC proliferation in the subventricular zone and migration towards the stroke-injured neocortex. Whether this response is sustained in the irradiated brain remains unknown. Here, we demonstrate that cranial IR in mice at an early postnatal age significantly reduced the number to neuronal progenitors responding to cortical stroke in adults. This was accompanied by a reduced number of microglia/macrophages in the peri-infarct cortex; however, the astrocytic response was not altered. Our findings indicate that IR impairs the endogenous repair capacity in the brain in response to stroke, hence pointing to another side effect of cranial radiotherapy which requires further attention.

A direct action of thyrotropin (TSH, thyroid-stimulating hormone) on bone precursors in humans is controversial. Studies in rodent models have provided conflicting findings. We used cells derived from a moderately differentiated osteosarcoma stably overexpressing human TSH receptors (TSHRs) as a model of osteoblast precursors (U2OS-TSHR cells) to investigate TSHR-mediated effects in bone differentiation in human cells. We review our findings that (1) TSHR couples to several different G proteins to induce upregulation of genes associated with osteoblast activity-interleukin 11 (IL-11), osteopontin (OPN), and alkaline phosphatase (ALPL) and that the kinetics of the induction and the G protein-mediated signaling pathways involved were different for these genes; (2) TSH can stimulate β-arrestin-mediated signal transduction and that β-arrestin 1 in part mediates TSH-induced pre-osteoblast differentiation; and (3) TSHR/insulin-like growth factor 1 (IGF1) receptor (IGF1R) synergistically increased OPN secretion by TSH and IGF1 and that this crosstalk was mediated by physical association of these receptors in a signaling complex that uses β-arrestin 1 as a scaffold. These findings were complemented using a novel β-arrestin 1-biased agonist of TSHR. We conclude that TSHR can signal via several transduction pathways leading to differentiation of this model system of human pre-osteoblast cells and, therefore, that TSH can directly regulate these bone cells., (Copyright © 2020 Boutin, Gershengorn and Neumann.)

Thyrotropin hormone (TSH) was reported to exhibit biphasic regulation of cAMP production in human thyroid slices; specifically, upregulation at low TSH doses transitioning to inhibition at high doses. We observed this phenomenon in HEK293 cells overexpressing TSH receptors (TSHRs) but in only 25% of human thyrocytes (hThyros) in vitro . Because TSHR expression in hThyros in vitro was low, we tested the hypothesis that high, in situ levels of TSHRs were needed for biphasic cAMP regulation. We increased expression of TSHRs by infecting hThyros with adenoviruses expressing human TSHR (AdhTSHR), measured TSH-stimulated cAMP production and TSHR homodimerization. TSHR mRNA levels in hThyros in vitro were 100-fold lower than in human thyroid tissue. AdhTSHR infection increased TSHR mRNA expression to levels found in thyroid tissue and flow cytometry showed that cell-surface TSHRs increased more than 15-fold. Most uninfected hThyro preparations exhibited monotonic cAMP production. In contrast, most hThyro preparations infected with AdhTSHR expressing TSHR at in vivo levels exhibited biphasic TSH dose responses. Treatment of AdhTSHR-infected hThyros with pertussis toxin resulted in monotonic dose response curves demonstrating that lower levels of cAMP production at high TSH doses were mediated by G i /G o proteins. Proximity ligation assays confirmed that AdhTSHR infection markedly increased the number of TSHR homodimers. We conclude that in situ levels of TSHRs as homodimers are needed for hThyros to exhibit biphasic TSH regulation of cAMP production., (Copyright © 2020 Boutin, Krieger, Marcus-Samuels, Klubo-Gwiezdzinska, Neumann and Gershengorn.)

The thyrotropin (TSH) receptor (TSHR) signals via G proteins of all four classes and β -arrestin 1. Stimulation of TSHR leads to increasing cAMP production that has been reported as a monotonic dose-response curve that plateaus at high TSH doses. In HEK 293 cells overexpressing TSHRs (HEK-TSHR cells), we found that TSHR activation exhibits an "inverted U-shaped dose-response curve" with increasing cAMP production at low doses of TSH and decreased cAMP production at high doses (>1 mU/ml). Since protein kinase A inhibition by H-89 and knockdown of β -arrestin 1 or β -arrestin 2 did not affect the decreased cAMP production at high TSH doses, we studied the roles of TSHR downregulation and of G i /G o proteins. A high TSH dose (100 mU/ml) caused a 33% decrease in cell-surface TSHR. However, because inhibiting TSHR downregulation with combined expression of a dominant negative dynamin 1 and β -arrestin 2 knockdown had no effect, we concluded that downregulation is not involved in the biphasic cAMP response. Pertussis toxin, which inhibits activation of G i /G o , abolished the biphasic response with no statistically significant difference in cAMP levels at 1 and 100 mU/ml TSH. Concordantly, co-knockdown of G i /G o proteins increased cAMP levels stimulated by 100 mU/ml TSH from 55% to 73% of the peak level. These data show that biphasic regulation of cAMP production is mediated by G s and G i /G o at low and high TSH doses, respectively, which may represent a mechanism to prevent overstimulation in TSHR-expressing cells. SIGNIFICANCE STATEMENT: We demonstrate biphasic regulation of TSH-mediated cAMP production involving coupling of the TSH receptor (TSHR) to Gs at low TSH doses and to G i/o at high TSH doses. We suggest that this biphasic cAMP response allows the TSHR to mediate responses at lower levels of TSH and that decreased cAMP production at high doses may represent a mechanism to prevent overstimulation of TSHR-expressing cells. This mechanism could prevent chronic stimulation of thyroid gland function., (U.S. Government work not protected by U.S. copyright.)

Thyroid stimulating hormone and insulin-like growth factor 1 receptors (TSHRs and IGF1Rs, respectively) interact leading to additive or synergistic stimulation of cellular responses. Recent findings provide evidence that the interaction between TSHRs and IGF1Rs is similar to that described for other G protein-coupled receptors and receptor tyrosine kinases. These types of interactions occur at or proximal to the receptors and are designated "receptor cross-talk." Herein, we describe our studies in human thyrocytes, human retro-orbital fibroblasts from Graves' orbitopathy patients and a model cell line that support the concept of TSHR/IGF1R cross-talk. We also discuss how receptor cross-talk is involved in stimulation by a monoclonal TSHR-stimulating antibody and how targeting both receptors may lead to novel treatments of Graves' orbitopathy.

Recently, we showed that TSH-enhanced differentiation of a human preosteoblast-like cell model involved a β -arrestin 1 ( β -Arr 1)-mediated pathway. To study this pathway in more detail, we sought to discover a small molecule ligand that was functionally selective toward human TSH receptor (TSHR) activation of β -Arr 1. High-throughput screening using a cell line stably expressing mutated TSHRs and mutated β -Arr 1 (DiscoverX1 cells) led to the discovery of agonists that stimulated translocation of β -Arr 1 to the TSHR, but did not activate G s -mediated signaling pathways, i.e., cAMP production. D3- β Arr (NCGC00379308) was selected. In DiscoverX1 cells, D3- β Arr stimulated β -Arr 1 translocation with a 5.1-fold greater efficacy than TSH and therefore potentiated the effect of TSH in stimulating β -Arr 1 translocation. In human U2OS-TSHR cells expressing wild-type TSHRs, which is a model of human preosteoblast-like cells, TSH upregulated the osteoblast-specific genes osteopontin (OPN) and alkaline phosphatase (ALPL). D3- β Arr alone had only a weak effect to upregulate these bone markers, but D3- β Arr potentiated TSH-induced upregulation of ALPL and OPN mRNA levels 1.6-fold and 5.5-fold, respectively, at the maximum dose of ligands. Furthermore, the positive allosteric modulator effect of D3- β Arr resulted in an increase of TSH-induced secretion of OPN protein. In summary, we have discovered the first small molecule positive allosteric modulator of TSHR. As D3- β Arr potentiates the effect of TSH to enhance differentiation of a human preosteoblast in an in vitro model, it will allow a novel experimental approach for probing the role of TSH-induced β -Arr 1 signaling in osteoblast differentiation., (U.S. Government work not protected by U.S. copyright.)

In order to study functions of normal human thyrocytes, we developed a protocol to obtain these cells in primary culture. Thyrocytes are obtained from normal tissue obtained at surgery for removal of thyroid neoplasms. Under sterile conditions, specimens are minced into small pieces, mono-dispersed cells are generated by digestion with collagenase type IV and the cells plated in tissue culture grade dishes in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). After 24 h of incubation at 37 °C in a humidified 5% CO 2 incubator, the supernatant containing non-adherent cells is removed and the adherent cells are propagated in DMEM with 10% FBS, 100 IU/mL penicillin, and 10 μg/mL streptomycin. Cells proliferate with a doubling time of 72-94 h and retain functional characteristics for 9-12 doublings. We have used them successfully in studies to elucidate the signaling by thyrotropin (TSH) and insulin-like growth factor 1.

It has been shown that the TSH receptor (TSHR) couples to a number of different signaling pathways, although the Gs-cAMP pathway has been considered primary. Here, we measured the effects of TSH on bone marker mRNA and protein expression in preosteoblast-like U2OS cells stably expressing TSHRs. We determined which signaling cascades are involved in the regulation of IL-11, osteopontin (OPN), and alkaline phosphatase (ALPL). We demonstrated that TSH-induced up-regulation of IL-11 is primarily mediated via the Gs pathway as IL-11 was up-regulated by forskolin (FSK), an adenylyl cyclase activator, and inhibited by protein kinase A inhibitor H-89 and by silencing of Gαs by small interfering RNA. OPN levels were not affected by FSK, but its up-regulation was inhibited by TSHR/Gi-uncoupling by pertussis toxin. Pertussis toxin decreased p38 MAPK kinase phosphorylation, and a p38 inhibitor and small interfering RNA knockdown of p38α inhibited OPN induction by TSH. Up-regulation of ALPL expression required high doses of TSH (EC50 = 395nM), whereas low doses (EC50 = 19nM) were inhibitory. FSK-stimulated cAMP production decreased basal ALPL expression, whereas protein kinase A inhibition by H-89 and silencing of Gαs increased basal levels of ALPL. Knockdown of Gαq/11 and a protein kinase C inhibitor decreased TSH-stimulated up-regulation of ALPL, whereas a protein kinase C activator increased ALPL levels. A MAPK inhibitor and silencing of ERK1/2 inhibited TSH-stimulated ALPL expression. We conclude that TSH regulates expression of different bone markers via distinct signaling pathways.

Cortical ischemia induces proliferation of neural stem/progenitor cells (NSPCs) in the subventricular zone (SVZ) and provokes migration of these cells toward the injured area. Despite sustained migration of NSPCs for an extended period of time after injury, they do not appear to survive. Here, we hypothesized that the anti-apoptotic broad-spectrum caspase inhibitor Q-VD-OPh would increase NSPC survival in the injured cortex. However, contrary to our expectations, caspase inhibition did not promote NSPC survival and cortical neurogenesis. On the contrary, it abolished ischemia-induced proliferation and decreased the number of migrating neuroblasts in the injured cortex. Moreover, caspase inhibition decreased the levels of the chemoattractant chemokine CCL2 (MCP-1) and the pro-inflammatory cytokine IL-1β. We hence for the first time show that caspase inhibition abrogates the response of NSPCs to an ischemic injury, presumably by inhibiting the production of pro-inflammatory factors. Thus, caution is warranted if anti-apoptotic strategies are applied for neuroprotection.

Context: There is no pathogenetically linked medical therapy for Graves' ophthalmopathy (GO). Lack of animal models and conflicting in vitro studies have hindered the development of such therapy. Recent reports propose that Graves' Igs bind to and activate thyrotropin receptors (TSHRs) and IGF-1 receptors (IGF-1Rs) on cells in orbital fat, stimulating hyaluronan (HA) secretion, a component of GO., Objective: The objective of the study was to investigate potential cross talk between TSHRs and IGF-1Rs in the pathogenesis of GO using a sensitive HA assay., Design/setting/participants: Orbital fibroblasts from GO patients were collected in an academic clinical practice and cultured in a research laboratory. Cells were treated with TSH, IGF-1, and a monoclonal Graves' Ig M22., Main Outcome Measures: HA was measured by a modified ELISA., Results: Simultaneous activation by TSH and IGF-1 synergistically increased HA secretion from 320 ± 52 for TSH and 430 ± 65 μg/mL for IGF-1 alone, to 1300 ± 95 μg/mL. IGF-1 shifted the TSH EC50 19-fold to higher potency. The dose response to M22 was biphasic. An IGF-1R antagonist inhibited the higher potency phase but had no effect on the lower potency phase. M22 did not cause IGF-1R autophosphorylation. A TSHR antagonist abolished both phases of M22-stimulated HA secretion., Conclusions: M22 stimulation of HA secretion by GO fibroblasts/preadipocytes involves cross talk between TSHR and IGF-1R. This cross talk relies on TSHR activation rather than direct activation of IGF-1R and leads to synergistic stimulation of HA secretion. These data propose a model for GO pathogenesis that explains previous contradictory results and argues for TSHR as the primary therapeutic target for GO.

To study the network dynamics of the riboflavin biosynthesis pathway and to identify potential bottlenecks in the system, an ordinary differential equation-based model was constructed using available literature data for production strains. The results confirmed that the RibA protein is rate limiting in the pathway. Under the conditions investigated, we determined a potential limiting order of the remaining enzymes under increased RibA concentration (>0.102 mM) and therefore higher riboflavin production (>0.045 mmol g(CDW)(-1) h(-1) and 0.0035 mM s(-1), respectively). The reductase activity of RibG and lumazine synthase (RibH) might be the next most limiting steps. The computational minimization of the enzyme concentrations of the pathway suggested the need for a greater RibH concentration (0.251 mM) compared with the other enzymes (RibG: 0.188 mM, RibB: 0.023 mM).

Because the TSH receptor (TSHR) plays an important role in the pathogenesis of thyroid disease, a TSHR antagonist could be a novel treatment. We attempted to develop a small molecule, drug-like antagonist of TSHR signaling that is selective and active in vivo. We synthesized NCGC00242364 (ANTAG3) by chemical modification of a previously reported TSHR antagonist. We tested its potency, efficacy, and selectivity in a model cell system in vitro by measuring its activity to inhibit stimulation of cAMP production stimulated by TSH, LH, or FSH. We tested the in vivo activity of ANTAG3 by measuring its effects to lower serum free T4 and thyroid gene expression in female BALB/c mice continuously treated with ANTAG3 for 3 days and given low doses of TRH continuously or stimulated by a single administration of a monoclonal thyroid-stimulating antibody M22. ANTAG3 was selective for TSHR inhibition; half-maximal inhibitory doses were 2.1 μM for TSHR and greater than 30 μM for LH and FSH receptors. In mice treated with TRH, ANTAG3 lowered serum free T4 by 44% and lowered mRNAs for sodium-iodide cotransporter and thyroperoxidase by 75% and 83%, respectively. In mice given M22, ANTAG3 lowered serum free T4 by 38% and lowered mRNAs for sodium-iodide cotransporter and thyroperoxidase by 73% and 40%, respectively. In conclusion, we developed a selective TSHR antagonist that is effective in vivo in mice. This is the first report of a small-molecule TSHR antagonist active in vivo and may lead to a drug to treat Graves' disease.

The TSH receptor (TSHR) is a member of the glycoprotein hormone receptors, a subfamily of family A G protein-coupled receptors. The TSHR is of great importance for the growth and function of the thyroid gland. The TSHR and its endogenous ligand TSH are pivotal proteins with respect to a variety of physiological functions and malfunctions. The molecular events of TSHR regulation can be summarized as a process of signal transduction, including signal reception, conversion, and amplification. The steps during signal transduction from the extra- to the intracellular sites of the cell are not yet comprehensively understood. However, essential new insights have been achieved in recent years on the interrelated mechanisms at the extracellular region, the transmembrane domain, and intracellular components. This review contains a critical summary of available knowledge of the molecular mechanisms of signal transduction at the TSHR, for example, the key amino acids involved in hormone binding or in the structural conformational changes that lead to G protein activation or signaling regulation. Aspects of TSHR oligomerization, signaling promiscuity, signaling selectivity, phenotypes of genetic variations, and potential extrathyroidal receptor activity are also considered, because these are relevant to an understanding of the overall function of the TSHR, including physiological, pathophysiological, and pharmacological perspectives. Directions for future research are discussed.

Context: Graves ophthalmopathy (GO) is an autoimmune disorder characterized by increased adipogenesis and hyaluronan (HA) production by orbital fibroblasts. Circulating autoantibodies (thyroid-stimulating antibodies [TSAbs]) directed at the thyrotropin receptor (TSHR) on these cells stimulate or augment these cellular processes. A recently developed drug-like small molecule inverse agonist of TSHR, NCGC00229600, termed 1, binds to TSHR and blocks basal and stimulated signal transduction., Objective: The purpose of this article was to determine whether 1 might inhibit HA production and relevant signaling pathways in orbital fibroblasts cultured in the presence of monoclonal TSAbs or bovine TSH (bTSH)., Design: Primary cultures of undifferentiated GO orbital fibroblasts (n = 13) were untreated or treated with a TSAb (M22 or MS-1) or bTSH in serum-free medium, with or without 1 or a TSHR neutral antagonist, NCGC00242595, termed 2, which does not inhibit basal signaling but does inhibit stimulated signaling., Main Outcome Measures: cAMP production, Akt phosphorylation (Ser473pAkt in media and immunoblotting for pAkt/total Akt), and HA production were analyzed., Results: Compound 1 inhibited basal cAMP, pAkt, and HA production and that stimulated by M22 in undifferentiated orbital fibroblasts. Inhibition of HA production was dose-dependent, with a half-maximal inhibitory dose of 830 nM. This compound also inhibited MS-1- and bTSH-stimulated cAMP, pAkt, and HA production. Compound 2 did not inhibit basal HA production but did inhibit M22-stimulated HA production., Conclusions: Because cAMP, pAkt, and HA production are fibroblast functions that are activated via TSHR signaling and are important in the pathogenesis of GO, small molecule TSHR antagonists may prove to be effective in the treatment or prevention of the disease in the future.

The physiological role of the TSH receptor (TSHR) as a major regulator of thyroid function is well understood, but TSHRs are also expressed in multiple normal extrathyroidal tissues, and the physiological roles of TSHRs in these tissues are unclear. Moreover, TSHRs play a major role in several pathological conditions including hyperthyroidism, hypothyroidism, and thyroid tumors. Small molecule, "drug-like" TSHR agonists, neutral antagonists, and inverse agonists may be useful as probes of TSHR function in extrathyroidal tissues and as leads to develop drugs for several diseases of the thyroid. In this Update, we review the most recent findings regarding the development and use of these small molecule TSHR ligands.

Context: Graves' disease (GD) is caused by persistent, unregulated stimulation of thyrocytes by thyroid-stimulating antibodies (TSAbs) that activate the TSH receptor (TSHR). We previously reported the first small-molecule antagonist of human TSHR and showed that it inhibited receptor signaling stimulated by sera from four patients with GD., Objective: Our objective was to develop a better TSHR antagonist and use it to determine whether inhibition of TSAb activation of TSHR is a general phenomenon., Design: We aimed to chemically modify a previously reported small-molecule TSHR ligand to develop a better antagonist and determine whether it inhibits TSHR signaling by 30 GD sera. TSHR signaling was measured in two in vitro systems: model HEK-EM293 cells stably overexpressing human TSHRs and primary cultures of human thyrocytes. TSHR signaling was measured as cAMP production and by effects on thyroid peroxidase mRNA., Results: We tested analogs of a previously reported small-molecule TSHR inverse agonist and selected the best NCGC00229600 for further study. In the model system, NCGC00229600 inhibited basal and TSH-stimulated cAMP production. NCGC00229600 inhibition of TSH signaling was competitive even though it did not compete for TSH binding; that is, NCGC00229600 is an allosteric inverse agonist. NCGC00229600 inhibited cAMP production by 39 ± 2.6% by all 30 GD sera tested. In primary cultures of human thyrocytes, NCGC00229600 inhibited TSHR-mediated basal and GD sera up-regulation of thyroperoxidase mRNA levels by 65 ± 2.0%., Conclusion: NCGC00229600, a small-molecule allosteric inverse agonist of TSHR, is a general antagonist of TSH receptor activation by TSAbs in GD patient sera.

Small molecule inverse agonists for the TSH receptor (TSHR) may be used as probes of the role of basal (or agonist-independent or constitutive) signaling and may have therapeutic potential as orally active drugs to inhibit basal signaling in patients with thyroid cancer and in some patients with hyperthyroidism. We describe the first small-molecule ligand [1;2-(3-((2,6-dimethylphenoxy)methyl)-4-methoxyphenyl)-3-(furan-2-ylmethyl)-2,3-dihydroquinazolin-4(1H)-one] that exhibits inverse agonist properties at TSHR. 1 inhibits basal and TSH-stimulated signaling, measured as cAMP production, by TSHRs in HEK-EM 293 cells stably expressing wild-type TSHRs; the antagonism of TSH-mediated signaling is competitive. 1 also inhibits basal signaling by wild-type TSHRs, and four constitutively active mutants of TSHR expressed transiently in HEK-EM 293 cells. 1 was active under more physiologically relevant conditions in primary cultures of human thyrocytes expressing endogenous TSHRs where it inhibited basal levels of mRNA transcripts for thyroglobulin, thyroperoxidase, sodium iodide symporter, and TSHR. These data serve as proof of principle that small, drug-like molecules can inhibit basal signaling by TSHR. We suggest that this small molecule is a lead compound for the development of higher-potency inverse agonists that can be used as probes of TSHR biology with therapeutic potential.

Receptors for thyrotropin-releasing hormone (TRH) and thyrotropin (thyroid-stimulating hormone-TSH) are important regulators of the function of the TSH-producing cells of the anterior pituitary gland and the thyroid gland, respectively, and thereby play a central role in thyroid hormone homeostasis. Although the roles of TRH- and TSH-stimulated signaling in these endocrine glands are well understood, these receptors are expressed in other sites and their roles in these extraglandular tissues are less well known. Moreover, one of the two subtypes of TRH receptors (TRH-R2) and the single TSH receptor (TSHR) exhibit constitutive signaling activity and the roles of constitutive signaling by these receptors are poorly understood. One approach to studying constitutive signaling is to use inverse agonists. In this chapter, we will describe the experimental procedures used to measure constitutive signaling by TRH-R2 and TSHR and the effects of their specific inverse agonists., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Seven-transmembrane-spanning receptors (7TMRs) are prominent drug targets. However, small-molecule ligands for 7-transmembrane-spanning receptors for which the natural ligands are large, heterodimeric glycoprotein hormones, like thyroid-stimulating hormone (TSH; thyrotropin), have only recently been reported, and none are approved for human use. We have used quantitative high-throughput screening to identify a small-molecule TSH receptor (TSHR) agonist that was modified to produce a second agonist with increased potency. We show that these agonists are highly selective for human TSHR versus other glycoprotein hormone receptors and interact with the receptor's serpentine domain. A binding pocket within the transmembrane domain was defined by docking into a TSHR homology model and was supported by site-directed mutagenesis. In primary cultures of human thyrocytes, both TSH and the agonists increase mRNA levels for thyroglobulin, thyroperoxidase, sodium iodide symporter, and deiodinase type 2, and deiodinase type 2 enzyme activity. Moreover, oral administration of the agonist stimulated thyroid function in mice, resulting in increased serum thyroxine and thyroidal radioiodide uptake. Thus, we discovered a small molecule that activates human TSHR in vitro, is orally active in mice, and could be a lead for development of drugs to use in place of recombinant human TSH in patients with thyroid cancer.

Low-molecular-weight (LMW) antagonists for TSH receptor (TSHR) may have therapeutic potential as orally active drugs to block stimulating antibodies (TsAbs) in Graves' hyperthyroidism. We describe an approach to identify LMW ligands for TSHR based on Org41841, a LMW partial agonist for the LH/choriogonadotropin receptor and TSHR. We used molecular modeling and functional experiments to guide the chemical modification of Org41841. We identified an antagonist (NIDDK/CEB-52) that selectively inhibits activation of TSHR by both TSH and TsAbs. Whereas initially characterized in cultured cells overexpressing TSHRs, the antagonist was also active under more physiologically relevant conditions in primary cultures of human thyrocytes expressing endogenous TSHRs in which it inhibited TSH- and TsAb-induced up-regulation of mRNA transcripts for thyroperoxidase. Our results establish this LMW compound as a lead for the development of higher potency antagonists and serve as proof of principle that LMW ligands that target TSHR could serve as drugs in patients with Graves' disease.

The thyroid-stimulating hormone (TSH; thyrotropin) receptor belongs to the glycoprotein hormone receptor subfamily of 7-transmembrane spanning receptors. TSH receptor (TSHR) is expressed mainly in thyroid follicular cells and is activated by TSH, which regulates the growth and function of thyroid follicular cells. Recombinant TSH is used in diagnostic screens for thyroid cancer, especially in patients after thyroid cancer surgery. Currently, no selective small-molecule agonists of the TSHR are available. To screen for novel TSHR agonists, the authors miniaturized a commercially available cell-based cyclic adenosine 3',5' monophosphate (cAMP) assay into a 1536-well plate format. This assay uses an HEK293 cell line stably transfected with the TSHR coupled to a cyclic nucleotide gated ion channel as a biosensor. From a quantitative high-throughput screen of 73,180 compounds in parallel with a parental cell line (without the TSHR), 276 primary active compounds were identified. The activities of the selected active compounds were further confirmed in an orthogonal homogeneous time-resolved fluorescence cAMP-based assay. Forty-nine compounds in several structural classes have been confirmed as the small-molecule TSHR agonists that will serve as a starting point for chemical optimization and studies of thyroid physiology in health and disease.

The selectivity of G-protein recognition is determined by the intracellular loops (ICLs) of seven-transmembrane-spanning receptors. In a previous study, we have shown that the N-terminal and central portions of ICL2 from F525 to D530 participate in dual Galphas-/Galphaq-protein activation by the thyroid-stimulating hormone receptor (TSHR). ICL3 is another major determinant for G-protein activation. Therefore, the aim of our study was to identify important amino acids within ICL3 of the TSHR to gain insight in more detail about its specific function for Galphas- and Galphaq-protein activation and selectivity. Single-alanine substitutions of residues in the N-terminal, middle, and C-terminal region of ICL3 were generated. N-terminal residues Y605 and V608 and C-terminal positions K618, K621, and I622 were identified as selectively important for Galphaq activation, whereas mutations in the center of ICL3 had no effect on TSHR signaling. Our findings provide evidence for an amino acid pattern in the N- and C-terminal part of ICL3, which is involved in Galphaq-mediated signaling. Furthermore, molecular modeling of interaction of TSHR ICL2 and 3 with Galphaq suggests three potential contact sites: TSHR C-terminal ICL3 with beta5-6 loop of Galphaq, TSHR ICL2 residues I523-R531 with beta2-3 loop and N-terminal helix of Galphaq, and TSHR ICL2/transmembrane helix (TMH) 3+ICL3/TMH6 with C-terminal tail of Galphaq.

The substituted thieno[2,3-d]pyrimidine 3 (Org 41841), a partial agonist for the luteinizing hormone/choriogonadotropin receptor (LHCGR) and the closely related thyroid-stimulating hormone receptor (TSHR), was fundamentally altered, and the resulting analogues were analyzed for their potencies, efficacies, and specificities at LHCGR and TSHR. Chemical modification of the parent compound combined with prior mutagenesis of TSHR provided compelling experimental evidence in support of computational models of 3 binding to TSHR and LHCGR within their transmembrane cores. Biochemical analysis of a specific modification to the chemical structure of 3 provides additional evidence of a H-bond between the ligand and a glutamate residue in transmembrane helix 3, which is conserved in both receptors. Several key interactions were surveyed to determine their respective biochemical roles in terms of both van der Waals dimensions and hydrogen bond capacity and the respective relationship to biological activity.

Constitutively-activating germline mutations of the thyrotropin receptor (TSHR) gene are very rare and are considered the cause of hereditary nonautoimmune hyperthyroidism. We describe four affected individuals from a Caucasian family: a mother and her three children, and an unaffected father. The mother and her first two children presented in a similar manner: lifelong histories of heat intolerance, hyperactivity, fast heart rate, reduced energy, increased appetite, and scrawny build. They all developed goiter in childhood and showed a suppressed TSH and elevated thyroxine (T(4)). The last child, a 12-year-old female, presented with no clinical symptoms or palpable neck mass, but with a suppressed TSH, elevated T(4) and thyromegaly detected by ultrasound. Mutation analysis of the TSHR gene in all family members revealed a novel heterozygous germline mutation resulting in the substitution of phenylalanine (TTC) by serine (TCC) at codon 631 in transmembrane helix 6 in the mother and all three children. Functional characterization of this germline mutation showed constitutive activation of the G(s)-mediated cyclic adenosine monophosphate (cAMP) pathway, which controls thyroid hormone production and thyroid growth. Molecular characterization of F631S demonstrates that this activating mutation plays a key role in the development of hereditary hyperthyroidism in this family although the timing of onset of clinical manifestations in the subjects may depend on other, as yet unidentified, factors.

Previous reports on the follicle-stimulating hormone receptor and choriogonadotropic/LH receptor, which belong to the glycoprotein hormone receptor family, suggest that the extracellular loop (ECL) 3 could be a key domain for ligand binding and intramolecular receptor signaling. In contrast to ECLs 1 and 2 of glycoprotein hormone receptors, the ECL3 displays high sequence homology, particularly in the central portion of the loop. Therefore, we opted to identify amino acids with functional importance within ECL3 of the TSH receptor (TSHR). Single alanine substitutions of all residues in ECL3 were generated. Functional characterization revealed the importance of five amino acids in the central portion of ECL3 and K660 at the ECL3/transmembrane helix (TMH) 7 junction for TSHR signaling. Decrease of G(s) activation and loss of G(q) activation by substitutions of K660 demonstrates a role for this position for TSHR conformation and signal transduction. By molecular modeling, we predicted potential interaction partners of K660:E409 and D410 in the N terminus of TMH1 and D573 in the ECL2. Complementary double mutants did not reconstitute G(s)/G(q)-mediated signaling, suggesting that K660 is not directly involved in a structural unit between ECL3 and the N terminus of TMH1. These results support a TSHR model in which the side chain of K660 is orientated toward the backbone of ECL2. Moreover, our findings provide evidence that a hydrophobic cluster, comprising residues 652-656 of ECL3, strongly influences intramolecular signal transduction and G protein activation of the TSHR.

The purpose of this review is to summarize current knowledge of the etiology of euthyroid and toxic multinodular goiter (MNG) with respect to the epidemiology, clinical characteristics, and molecular pathology. In reconstructing the line of events from early thyroid hyperplasia to MNG we will argue the predominant neoplastic character of nodular structures, the nature of known somatic mutations, and the importance of mutagenesis. Furthermore, we outline direct and indirect consequences of these somatic mutations for thyroid pathophysiology and summarize information concerning a possible genetic background of euthyroid goiter. Finally, we discuss uncertainties and open questions in differential diagnosis and therapy of euthyroid and toxic MNG.

The TSH receptor (TSHR) activates mainly two signal transduction pathways, cAMP production and phosphoinositide turnover, mediated by Gs and Gq coupling, respectively. Several activating deletion and point mutations within intracellular loop 3 (ICL3) and the adjacent portion of transmembrane domain 6 (TM6) support a direct G protein activation by this receptor domain. The ICL3, however, is predicted by modeling to interact with other receptor domains, primarily ICL2, to form a pocket for G protein binding and to allow optimum interaction. Systematic mutagenesis was used to identify important sites within ICL2 and potential interactions between ICL2 and ICL3 of the TSHR required for G protein coupling. Deletions of four or five residues and their corresponding multiple alanine substitutions were introduced into ICL2. Residues I523-D530, comprising mainly the N-terminal half of ICL2, appeared to be critical for Gs- and Gq-mediated signaling. A single alanine substitution screening within ICL2 revealed hydrophobic residue M527 in particular and, to lesser extents, F525, R528, L529, and D530 as residues that selectively abolished or strongly impaired Gq activation. Molecular modeling suggests that F525 interacts with ICL3. To test this hypothesis, ICL2/ICL3 double mutants introducing strong complementary properties were constructed and tested for functional rescue of Gq-mediated signaling. Our results indicate that ICL2 interacts with ICL3 in close vicinity to F525 and T607, suggesting a conformational cooperation between ICL2 and ICL3 during Gq activation by TSHR.

Euthyroid goiter is characterized by diffuse or nodular enlargement of the thyroid gland. Iodine deficiency and cigarette smoking have been identified as important environmental factors. However, family and twin pair studies suggest a strong genetic predisposition. Therefore, we performed the first extended genome-wide scan to identify susceptibility loci that predispose for euthyroid goiter using 450 microsatellite markers in 18 extended Danish, German, and Slovakian families. Parametric and nonparametric multipoint linkage analyses were performed. The highest nonparametric LOD scores were obtained for chromosomes 2q and 3p with values of 2.54 at D2S1363 and 2.25 at D3S3038, respectively. Assuming heterogeneity and dominant inheritance, heterogeneity LOD scores (HLOD) of 2.71 and 1.94 were calculated for 2q and 3p, respectively. Furthermore, nonparametric LOD scores of 1.87 (HLOD 1.39) at D7S1808 on 7q and 1.79 (HLOD 1.80) at D8S264 on 8p were obtained. Haplotyping of families contributing to the linkage signals revealed four families compatible with a putative locus on 3p and one family each showing strict cosegregation with the loci on 2q, 7q, and 8p. The four novel candidate loci corroborate the assumed heterogeneity in the etiology of euthyroid familial goiter. For the first time, a more prevalent putative locus, present in 20% of the families investigated, was identified.

Objective: To describe a kindred with a rare RET germline mutation in codon 791 and discuss potential management strategies., Methods: We present clinical and biochemical data as well as results of mutation analysis in our study subjects and provide an overview of related published reports., Results: Multiple endocrine neoplasia type 2 (MEN 2) is a familial cancer syndrome characterized by the development of medullary thyroid carcinoma (MTC), pheochromocytoma, and parathyroid hyperplasia or adenoma. Germline mutations in RET are responsible for this autosomal dominant syndrome. Familial MTC is a variant of MEN 2A and can be caused by RET mutations in codon 791. Deaths from gene carriers with mutations in these codons have not yet been reported. In general, gene carriers with these RET mutations have late-onset MTC. Because only a few kindreds with this specific mutation have been identified and no long-term follow-up data are available, management of these patients can be a challenge. We illustrate the difficulties with decisions about not only when to perform thyroidectomy in these patients but also whether thyroidectomy should even be considered in such gene carriers with a benign course. Our reported kindred included four carriers with a codon 791 RET germline mutation, one of whom had the rare concomitant occurrence of acromegaly and MEN 2A. The 70-year-old mother had acromegaly and hyperparathyroidism but normal serum calcitonin levels and normal findings on thyroid ultrasound examination. She refused pentagastrin testing and any surgical intervention. The 37-year-old daughter had hypothyroidism, a small thyroid gland, and negative results of pentagastrin stimulation testing of calcitonin. The 18-year-old grandson also had a negative pentagastrin test result and normal thyroid ultrasound findings. The 5-year-old granddaughter had normal results of thyroid ultrasonography. In all patients, we recommended thyroidectomy., Conclusion: Prospective studies are needed to clarify which patients with codon 791 RET germline mutation should undergo thyroidectomy.

Iodine deficiency is the most important etiological factor for euthyroid endemic goiter. However, family and twin pair studies also strongly indicate a genetic prediposition. In euthyroid goiters molecular defects in the thyroglobulin (TG), and Na+/I- symporter (NIS) gene have been identified. Numerous mutations in the Pendrin (PDS) gene have been found in families with PDS characterized by deafness and euthyroid goiter. Moreover, family studies indicated two major candidate loci MNG-1 on chromosome 14q31 and Xp22. However, all previous linkage studies investigated only one family. To clarify the general relevance of these previously identified two major candidate loci for the etiology of euthyroid goiter we investigated four families with a total number of 74 family members by linkage analysis with microsatellite markers. Moreover, we analyzed the thyroid candidate genes TG, thyroperoxidase (TPO), NIS, TSH receptor, and PDS. In a further family with 12 members in whom we have previously demonstrated linkage to the MNG-1 locus we investigated the Xp22 locus and the PDS gene in addition to our initial study. Linkage analysis results of our study are not significant enough to definitely exclude or confirm linkage to the investigated candidate genes and loci. Nevertheless, we obtained very weak indications for possible linkage to Xp22 in one family by a maximal multipoint LOD score of 1.15, and cosegregation of haplotypes among affected family members. Moreover, in another family linkage to PDS was indicated by a maximal multipoint LOD score of 1.87 as well as cosegregation of haplotypes. However, sequencing of the PDS gene did not reveal germline mutations. A significant total NPL score of 6.5 for PDS over all families most likely indicated linkage to a genomic region close to PDS. Furthermore, the likelihood of linkage to MNG-1 and Xp22 is reduced, because multipoint LOD scores were below 1 or negative. In all families there was no significant evidence for linkage for the thyroid candidate genes TG, TPO, NIS, or the TSH receptor. In conclusion, a general role of MNG-1 and Xp22 for the etiology of euthyroid goiter is unlikely but cannot clearly excluded. The multipoint parametric and nonparametric LOD scores further suggest genetic heterogeneity in the etiology of familial euthyroid goiter. To identify other susceptibility loci it is necessary to perform genome-wide linkage analysis studies with more families.