Graves hyperthyroidism (GH) is a condition in which autoantibodies chronically activate the thyrotropin (TSH) receptor (TSHR). TSHR is one of the few G protein-coupled receptors (GPCRs) predicted to have a signal peptide, making it a potential target for cyclotriazadisulfonamide (CADA) compounds. We sought to determine whether a small-molecule drug that selectively induces nascent protein degradation could decrease TSHR expression in vitro and in vivo at therapeutically relevant levels. We tested several CADA compounds for their ability to reduce TSHR surface expression in HEK 293 cells overexpressing human TSHR (HEK-TSHR cells) using flow cytometry. Inhibition of downstream cAMP production and thyroglobulin (Tg) secretion were measured in HEK-TSHR and human thyrocytes, respectively. Follow-up studies in VGD040-treated BALB/c mice assessed plasma levels of free T4 in response to TSH stimulation. Among a number of CADA analogues, VGD040 decreased TSHR at the surface of HEK-TSHR cells. VGD040 was found to be selective toward TSHR compared to similar glycoprotein hormone receptors. In human thyrocytes, reduction of TSHR surface expression by VGD040 decreased cyclic adenosine monophosphate production and Tg secretion. Most important, VGD040 decreased TH secretion in mice without apparent toxicity at the effective dose studied. VGD040 is an important new lead with potential for developing safe drug treatments for GH.

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.

Background: Thyrotropin receptor (TSHR) and insulin-like growth factor 1 receptor (IGF-1R) have been shown to crosstalk in primary cultures of human thyrocytes (hThyros) and Graves' orbital fibroblasts. The phenomenon of TSHR/IGF-1R crosstalk has been largely studied in the pathogenesis of thyroid eye disease (TED) in human orbital fibroblasts. Here, we investigated the effects of inhibiting the IGF-1R-mediated contribution to crosstalk by linsitinib (Lins), a small-molecule IGF-1R kinase inhibitor, on TSH-induced regulation of thyroperoxidase (TPO) and thyroglobulin (TG) mRNAs and proteins in hThyros in vitro, and on TPO and TG mRNAs and free thyroxine (fT4) levels in vivo in mice. Methods: Steady-state levels of mRNAs of TPO and TG in hThyros in vitro and mouse thyroid glands were measured by RT-qPCR. Human TG (hTG) and human TPO (hTPO) proteins in human thyroid cell cultures were measured by Western blot or ELISA. Translation rates of hTG were quantified by stable isotope labeling by amino acids method (SILAC). Thyroidal mouse Tpo (mTpo) and Tg (mTg) mRNAs and fT4 in mice were assessed after Lins administration on 3 consecutive days followed by an intraperitoneal dose of bovine TSH (bTSH) 3 hours prior to drawing blood. Results: In primary cultures of hThyros, Lins inhibited bTSH-induced upregulation of hTPO mRNA by 61.5%, and hTPO protein was inhibited by 42.4%. There was no effect of Lins on hTG mRNA, but Lins inhibited the upregulation of secreted and cell-associated hTG protein by 50.1% and 42.2%, respectively, by inhibiting hTG mRNA translation. mTpo mRNA measured in thyroid glands after treatment with Lins was reduced by 31.5%. There was no effect of Lins on mTg mRNA, however, Lins decreased fT4 levels in mice under basal (endogenous mTSH levels) and bTSH-treated conditions. Conclusions: The IGF-1R antagonist Lins inhibited bTSH-stimulated hTG and hTPO protein expression in primary cultures of hThyros and fT4 levels in mice. We suggest that thyroid function studies be monitored when Lins is administered to humans, for example, if it is used to treat TED.

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.

The TSH receptor (TSHR) is the major regulator of thyroid hormone biosynthesis in human thyrocytes by regulating the transcription of a number of genes including thyroglobulin (TG) and thyroperoxidase (TPO). Until recently, it was thought that TSHR initiated signal transduction pathways only at the cell-surface and that internalization was primarily involved in TSHR desensitization and downregulation. Studies primarily in mouse cells showed that TSHR internalization regulates gene transcription at an intracellular site also. However, this has not been shown for genes involved in thyroid hormone biosynthesis in human thyrocytes. We used human thyrocytes in primary culture. In these cells, the dose-response to TSH for gene expression is biphasic with low doses upregulating gene expression and higher doses decreasing gene expression. We used two approaches to inhibit internalization. In the first, we used inhibitors of dynamins, dynasore and dyngo-4a. Pretreatment with dynasore or dyngo-4a markedly inhibited TSH upregulation of TG and TPO mRNAs, as well as TG secretion. In the second, we used knockdown of dynamin 2, which is the most abundant dynamin in human thyrocytes. We showed that dynamin 2 knockdown inhibited TSHR internalization and decreased the TSH-stimulated levels of TG and TPO mRNAs and proteins. Lastly, we showed that the level of the activatory transcription factor phosphorylated cAMP response element binding protein (pCREB) in the cell nuclei was reduced by 68% when internalization was inhibited. We conclude that upregulation of genes involved in thyroid hormone synthesis in human thyrocytes is, in part, dependent on internalization leading to nuclear localization of an activated transcription factor(s).

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.

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) beta-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.

LettersOctober 2021Combined Immunosuppressive Therapy for Severe Graves DermopathySriram Gubbi, MD, Katherine Andrea Araque, MD, Shirisha Avadhanula, MD, Asma Azam, BS, Roxanne Merkel, RN, Craig Cochran, RN, Peter Denis Burbelo, PhD, Meryl Waldman, MD, Susanne Neumann, PhD, Marvin Carl Gershengorn, MD, Monica Skarulis, MD, and Joanna Klubo-Gwiezdzinska, MD, PhDSriram Gubbi, MDNational Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MarylandSearch for more papers by this author, Katherine Andrea Araque, MDNational Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MarylandSearch for more papers by this author, Shirisha Avadhanula, MDCleveland Clinic, Cleveland, OhioSearch for more papers by this author, Asma Azam, BSNational Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MarylandSearch for more papers by this author, Roxanne Merkel, RNNational Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MarylandSearch for more papers by this author, Craig Cochran, RNNational Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MarylandSearch for more papers by this author, Peter Denis Burbelo, PhDNational Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MarylandSearch for more papers by this author, Meryl Waldman, MDNational Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MarylandSearch for more papers by this author, Susanne Neumann, PhDNational Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MarylandSearch for more papers by this author, Marvin Carl Gershengorn, MDNational Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MarylandSearch for more papers by this author, Monica Skarulis, MDNational Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MarylandSearch for more papers by this author, and Joanna Klubo-Gwiezdzinska, MD, PhDNational Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MarylandSearch for more papers by this authorAuthor, Article, and Disclosure Informationhttps://doi.org/10.7326/L21-0152 SectionsAboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinkedInRedditEmail Background: The extrathyroidal manifestations of Graves disease include orbitopathy, dermopathy, and acropachy (periosteal changes and clubbing of fingers and toes). Severe forms of Graves dermopathy, such as nodular dermopathy or elephantiasis nostras verrucosa, can cause significant morbidity (1). The pathophysiology of Graves dermopathy is poorly understood. A combination of factors may contribute to its development, including immunologic factors, such as infiltration of T and B lymphocytes, cytokine production, interactions between serum thyroid-stimulating hormone receptor antibodies (TRAbs) and thyroid-stimulating hormone and insulin-like growth factor I receptors in dermal tissues; cellular factors, such as glycosaminoglycan production from fibroblasts; and other factors, such ...References1. Fatourechi V. Pretibial myxedema: pathophysiology and treatment options. Am J Clin Dermatol. 2005;6:295-309. [PMID:16252929] CrossrefMedlineGoogle Scholar2. Heyes C, Nolan R, Leahy M, et al. Treatment-resistant elephantiasic thyroid dermopathy responding to rituximab and plasmapheresis. Australas J Dermatol. 2012;53:e1-4. [PMID: 22309343] doi:10.1111/j.1440-0960.2010.00693.x CrossrefMedlineGoogle Scholar3. Kotwal A, Turcu AF, Sonawane V, et al. Clinical experience with rituximab and intravenous immunoglobulin for pretibial myxedema: a case series. Thyroid. 2019;29:692-699. [PMID: 30854936] doi:10.1089/thy.2018.0706 CrossrefMedlineGoogle Scholar4. Klubo-Gwiezdzinska J, Lange M, Cochran E, et al. Combined immunosuppressive therapy induces remission in patients with severe type B insulin resistance: A prospective cohort study. Diabetes Care. 2018;41:2353-2360. [PMID: 30201849] doi:10.2337/dc18-0884 CrossrefMedlineGoogle Scholar5. Farivar SS, Cunningham WE, Hays RD. Correlated physical and mental health summary scores for the SF-36 and SF-12 Health Survey, V.I. Health Qual Life Outcomes. 2007;5:54. [PMID: 17825096] CrossrefMedlineGoogle Scholar6. Oltulu P, Ince B, Kokbudak N, et al. Measurement of epidermis, dermis, and total skin thicknesses from six different body regions with a new ethical histometric technique. Turkish Journal of Plastic Surgery. 2018;26:56–61. doi:10.4103/tjps.TJPS_2_17 CrossrefGoogle Scholar Author, Article, and Disclosure InformationAffiliations: National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MarylandCleveland Clinic, Cleveland, OhioNational Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MarylandNote: The study was approved by the Institutional Board Review at the National Institutes of Health Clinical Center and an informed consent was obtained from the patient. Drs. Gubbi and Araque contributed equally to the study and preparation of the manuscript. Dr. Burbelo performed the luciferase immunoprecipitation systems assay. Dr. Gershengorn, Dr. Neumann, and Ms. Azam performed the in vitro studies. These in vitro studies are not available in clinical laboratories. Additional details about the tests used to establish the effectiveness of therapy are available from Dr. Klubo-Gwiezdzinska (e-mail, joanna.klubo-[email protected]gov).Acknowledgment: The authors thank the physicians and nurses who played a role in taking care of this patient at the National Institutes of Health Clinical Center.Financial Support: This patient was under the functional thyroid disease protocol at the National Institutes of Health, which was funded by the intramural program of the National Institutes of Health (DK 075005 16) and National Institute of Dental and Craniofacial Research. The sponsors had no role in the design or conduct of the study; collection, management, analysis, or interpretation of the data; preparation, review, or approval of the manuscript; or decision to submit the manuscript for publication.Disclosures: Authors have reported no disclosures of interest. Forms can be viewed at www.acponline.org/authors/icmje/ConflictOfInterestForms.do?msNum=L21-0152.Corresponding Author: Joanna Klubo-Gwiezdzinska, MD, PhD, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 10 Center Drive, Building 10 CRC, Room 9C-103, Bethesda, MD, 20814; e-mail, joanna.[email protected]gov.This article was published at Annals.org on 22 June 2021. PreviousarticleNextarticle Advertisement FiguresReferencesRelatedDetails Metrics October 2021Volume 174, Issue 10Page: 1478-1480KeywordsAntibodiesDisclosureImmunoprecipitationImmunosuppressivesInsulin resistanceMagnetic resonance imagingSoft tissuesThyroglobulinThyroid-stimulating hormoneThyroxine ePublished: 22 June 2021 Issue Published: October 2021 Copyright & PermissionsCopyright © 2021 by American College of Physicians. All Rights Reserved.PDF downloadLoading ...

Background: The pathogenesis of Graves' hyperthyroidism (GH) and associated Graves' orbitopathy (GO) appears to involve stimulatory autoantibodies (thyrotropin receptor [TSHR]-stimulating antibodies [TSAbs]) that bind to and activate TSHRs on thyrocytes and orbital fibroblasts. In general, measurement of circulating TSHR antibodies by clinical assays correlates with the status of GH and GO. However, most clinical measurements of TSHR antibodies use competitive binding assays that do not distinguish between TSAbs and antibodies that bind to but do not activate TSHRs. Moreover, clinical assays for TSAbs measure stimulation of only one signaling pathway, the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway, in engineered cells that are not thyrocytes or orbital fibroblasts. We determined whether measuring TSAbs by a cAMP-PKA readout in engineered cells accurately reveals the efficacies of stimulation by these antibodies on thyrocytes and orbital fibroblasts. Methods: We measured TSAb stimulation of normal human thyrocytes and orbital fibroblasts from patients with GO in primary cultures in vitro. In thyrocytes, we measured secretion of thyroglobulin (TG) and in orbital fibroblasts secretion of hyaluronan (hyaluronic acid [HA]). We also measured stimulation of cAMP production in engineered TSHR-expressing cells in an assay similar to clinical assays. Furthermore, we determined whether there were differences in stimulation of thyrocytes and orbital fibroblasts by TSAbs from patients with GH alone versus from patients with GO understanding that patients with GO have accompanying GH. Results: We found a positive correlation between TSAb stimulation of cAMP production in engineered cells and TG secretion by thyrocytes as well as HA secretion by orbital fibroblasts. However, TSAbs from GH patients stimulated thyrocytes more effectively than TSAbs from GO patients, whereas TSAbs from GO patients were more effective in activating orbital fibroblasts than TSAbs from GH patients. Conclusions: Clinical assays of stimulation by TSAbs measuring activation of the cAMP-PKA pathway do correlate with stimulation of thyrocytes and orbital fibroblasts; however, they do not distinguish between TSAbs from GH and GO patients. In vitro, TSAbs exhibit selectivity in activating TSHRs since TSAbs from GO patients were more effective in stimulating orbital fibroblasts and TSAbs from GH patients were more effective in stimulating thyrocytes.

Background Thyrotropin (TSH) and thyroid-stimulating antibodies (TSAbs) activate TSH receptor (TSHR) signaling by binding to its extracellular domain. TSHR signaling has been studied extensively in animal thyrocytes and in engineered cell lines, and differences in signaling have been observed in different cell systems. We, therefore, decided to characterize and compare TSHR signaling mediated by TSH and monoclonal TSAbs in human thyrocytes in primary culture. Methods We used quantitative RT-PCR to measure mRNA levels of thyroid specific genes thyroglobulin (TG), thyroperoxidase (TPO), iodothyronine deiodinase type 2 (DIO2), sodium-iodide symporter (NIS), and TSHR after stimulation by TSH or two monoclonal TSAbs, KSAb1and M22. We also compared secreted TG protein after TSHR activation by TSH and TSAbs using an ELISA. TSHR cell surface expression was determined using Fluorescence Activated Cell Sorting (FACS). Results We found that TSH at low doses increases and at high doses (> 1 mU/ml) decreases levels of gene expression for TSHR, TG, TPO, NIS, and DIO2. The biphasic effect of TSH on signaling was not caused by downregulation of cell surface TSHRs. This bell-shaped, biphasic dose-response curve has been termed an inverted U-shaped dose-response curve (IUDRC). An IUDRC was also found for TSH-induced regulation of TG secretion. In contrast, KSAb1- and M22-induced regulation of TSHR, TG, TPO, NIS, and DIO2 gene expression, and secreted TG followed a monotonic dose-response curve that plateaus at high doses of activating antibody. Conclusions Our data demonstrate that the physiological activation of TSHRs by TSH in primary cultures of human thyrocytes is characterized by a regulatory mechanism that may inhibit thyrocyte overstimulation. In contrast, TSAbs do not exhibit biphasic regulation. Although KSAb1 and M22 may not be representative of all TSAbs found in patients with Graves' disease, we suggest that persistent robust stimulation of TSHRs by TSAbs, unrelieved by a decrease at high TSAb levels, fosters chronic stimulation of thyrocytes in Graves' hyperthyroidism.

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 Gi/Go 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.

In contrast to most rhodopsin-like G protein-coupled receptors, the glycoprotein hormone receptors (GPHR) have a large extracellular N-terminus for hormone binding. The hormones do not directly activate the transmembrane domain but mediate their action via a, thus, far only partially knownTetheredAgonisticLIgand (TALI). The existence of such an intramolecular agonist was initially indicated by site-directed mutation studies and activating peptides derived from the extracellular hinge region. It is still unknown precisely howTALIis involved in intramolecular signal transmission. We combined systematic mutagenesis studies at the luteinizing hormone receptor and the thyroid-stimulating hormone receptor (TSHR), stimulation with a drug-like agonist (E2) of the TSHR, and structural homology modeling to unravel the functional and structural properties defining theTALIregion. Here, we report thatTALI(a) is predisposed to constitutively activate GPHR, (b) can by itself rearrange GPHR into a fully active conformation, (c) stabilizes active GPHR conformation, and (d) is not involved in activation of the TSHR by E2. In the active state conformation,TALIforms specific interactions between the N-terminus and the transmembrane domain. We show that stabilization of an active state is dependent onTALI,including activation by hormones and constitutively activating mutations.

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.

Increasing evidence of interdependence between G protein-coupled receptors and receptor tyrosine kinase signaling pathways has prompted reevaluation of crosstalk between these receptors in disease and therapy. Investigations into thyroid-stimulating hormone (TSH) and insulin-like growth factor 1 (IGF1) receptor crosstalk, and its application to the clinic have in particular shown recent progress. In this review, we summarize current insights into the mechanism of TSH/IGF1 receptor crosstalk. We discuss evidence that crosstalk is one of the underlying causes of TSHR-based disease and the feasibility of using combinations of TSH receptor and IGF1 receptor antagonists to increase the therapeutic index for the treatment of Graves' hyperthyroidism and Graves' ophthalmopathy.

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 Gi/Go 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 Gi/Go, abolished the biphasic response with no statistically significant difference in cAMP levels at 1 and 100 mU/ml TSH. Concordantly, co-knockdown of Gi/Go 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 Gs and Gi/Go 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 Gi/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.

In this review, we summarize the evidence against direct stimulation of insulin-like growth factor 1 receptors (IGF1Rs) by autoantibodies in Graves' orbitopathy (GO) pathogenesis. We describe a model of thyroid-stimulating hormone (TSH) receptor (TSHR)/IGF1R crosstalk and present evidence that observations indicating IGF1R's role in GO could be explained by this mechanism. We evaluate the evidence for and against IGF1R as a direct target of stimulating IGF1R antibodies (IGF1RAbs) and conclude that GO pathogenesis does not involve directly stimulating IGF1RAbs. We further conclude that the preponderance of evidence supports TSHR as the direct and only target of stimulating autoantibodies in GO and maintain that the TSHR should remain a major target for further development of a medical therapy for GO in concert with drugs that target TSHR/IGF1R crosstalk.

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.

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.

Endogenously expressed TSH receptors (TSHRs) on orbital fibroblasts of patients with Graves ophthalmopathy (GO) use crosstalk with IGF1 receptors (IGF1R) to synergistically stimulate secretion of hyaluronan (HA), a major component of GO pathology. We previously showed crosstalk occurred upstream of mitogen-activated protein kinase (ERK) phosphorylation. Because other G protein-coupled receptors engage arrestin-beta-1 (ARRB1) and ERK, we tested whether ARRB1 was a necessary component of TSHR/IGF1R crosstalk. HA secretion was stimulated by the TSHR-stimulating monoclonal antibodies M22 and KSAb1, or immunoglobulins from patients with GO (GO-Igs). Treatment with M22, as previously shown, resulted in biphasic dose-response stimulation of HA secretion. The high-potency phase was IGF1R dependent, and the low-potency phase was partly IGF1R independent. KSAb1 produced a monophasic dose-response stimulation of HA secretion, whose potency was lowered >20-fold after IGF1R knockdown. ARRB1 knockdown abolished M22's high-potency phase and lowered KSAb1's potency and efficacy. ARRB1 knockdown inhibited GO-Ig stimulation of HA secretion and of ERK phosphorylation. Last, ARRB1 was shown to be necessary for TSHR/IGF1R proximity. In contrast, ARRB2 knockdowns did not show these effects. Thus, TSHR must neighbor IGF1R for crosstalk in GO fibroblasts to occur, and this depends on ARRB1 acting as a scaffold. Similar scaffolding of TSHR and IGF1R by ARRB1 was found in human osteoblast-like cells and human thyrocytes. These findings support a model of TSHR/IGF1R crosstalk that may be a general mechanism for G-protein-coupled receptor/receptor tyrosine kinase crosstalk dependent on ARRB1.