Linsitinib Decreases Thyrotropin-Induced Thyroid Hormone Synthesis by Inhibiting Crosstalk Between Thyroid-Stimulating Hormone and Insulin-Like Growth Factor 1 Receptors in Human Thyrocytes and in Mice.

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 and on TPO and TG mRNAs and free thyroxine (fT4) levels in mice.

An Orally Efficacious Thyrotropin Receptor Ligand Inhibits Growth and Metastatic Activity of Thyroid Cancers.

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.

Opposing Effects of EGF Receptor Signaling on Proliferation and Differentiation Initiated by EGF or TSH/EGF Receptor Transactivation.

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.

Proximity ligation assay to study TSH receptor homodimerization and crosstalk with IGF-1 receptors in human thyroid cells.

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.

TSH stimulation of human thyroglobulin and thyroid peroxidase gene transcription is partially dependent on internalization.

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.

Inhibition of TSH/IGF-1 Receptor Crosstalk by Teprotumumab as a Treatment Modality of Thyroid Eye Disease.

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).

Graves' Autoantibodies Exhibit Different Stimulating Activities in Cultures of Thyrocytes and Orbital Fibroblasts Not Reflected by Clinical Assays.

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.

Targeting TSH and IGF-1 Receptors to Treat Thyroid Eye Disease.

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.

Thyrotropin regulation of differentiated gene transcription in adult human thyrocytes in primary culture.

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.

Is There Evidence for IGF1R-Stimulating Abs in Graves' Orbitopathy Pathogenesis?

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.

β-Arrestin 1 in Thyrotropin Receptor Signaling in Bone: Studies in Osteoblast-Like Cells.

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.

TSH Receptor Homodimerization in Regulation of cAMP Production in Human Thyrocytes .

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) . Because TSHR expression in hThyros was low, we tested the hypothesis that high, levels of TSHRs were needed for biphasic cAMP regulation.

TSH/IGF1 receptor crosstalk: Mechanism and clinical implications.

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.

Thyrotropin, but Not Thyroid-Stimulating Antibodies, Induces Biphasic Regulation of Gene Expression in Human Thyrocytes.

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.

Thyrotropin Causes Dose-dependent Biphasic Regulation of cAMP Production Mediated by G and G Proteins.

The thyrotropin (TSH) receptor (TSHR) signals via G proteins of all four classes and -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).

Arrestin-β-1 Physically Scaffolds TSH and IGF1 Receptors to Enable Crosstalk.

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-β-1 (ARRB1) and ERK, we tested whether ARRB1 was a necessary component of TSHR/IGF1R crosstalk.

Association of Thyrotropin Suppression With Survival Outcomes in Patients With Intermediate- and High-Risk Differentiated Thyroid Cancer.

Importance: Suppression of thyrotropin (often referred to as thyroid-stimulating hormone, or TSH) with levothyroxine used in management of intermediate- and high-risk differentiated thyroid cancer (DTC) to reduce the likelihood of progression and death is based on conflicting evidence.

Objective: To examine a cohort of patients with intermediate- and high-risk DTC to assess the association of thyrotropin suppression with progression-free survival (PFS) and overall survival.

Design, Setting, And Participants: This cohort study used a multicenter database analysis including patients from tertiary referral centers and local clinics followed up for a mean (SD) of 7.

Thyroid Stimulating Hormone (TSH)/Insulin-like Growth Factor 1 (IGF1) Receptor Cross-talk in Human Cells.

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.

Evaluation of the effects of chemotherapy-induced fatigue and pharmacological interventions in multiple mouse behavioral assays.

Fatigue is a common symptom in many diseases and disorders and can reduce quality of life, yet lacks an adequate pharmacological intervention. To identify and develop such interventions, and to better understand fatigue, additional preclinical research is necessary. However, despite numerous mouse behavioral assays reportedly detecting fatigue-like behavior, the assumption that fatigue-like behavior is detected in many assays has not been validated through a cross-assay study.

Normal Human Thyrocytes in Culture.

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).