Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Boutin A

PROVIDER: S-EPMC7203478 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Boutin Alisa A Krieger Christine C CC Marcus-Samuels Bernice B Klubo-Gwiezdzinska Joanna J Neumann Susanne S Gershengorn Marvin C MC

Frontiers in endocrinology 20200430

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 cAM ...[more]

PMID: 32425890

Similar Datasets

Project description: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 reverse transcription-polymerase chain reaction 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, KSAb1 and M22. We also compared secreted TG protein after TSHR activation by TSH and TSAbs using an enzyme-linked immunosorbent assay. 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.

Project description:PurposeFibrocytes (FC) are bone marrow-derived progenitor cells that are more abundant and infiltrate the thyroid and orbit in Graves orbitopathy (GO). FCs express high levels of thyrotropin receptor (TSHR) and insulin-like growth factor-1 receptor (IGF-1R). These receptors are physically and functionally associated, but their role in GO pathogenesis is not fully delineated. Treatment of FCs with thyroid stimulating hormone (TSH) or M22 (activating antibody to TSHR) induces the production of numerous cytokines, including tumor necrosis factor ? (TNF?). Teprotumumab (TMB) is a human monoclonal IGF-1R blocking antibody currently in clinical trial for GO and inhibits TSHR-mediated actions in FCs.AimTo characterize the molecular mechanisms underlying TSH-induced TNF? production by FCs, and the role of IGF-1R blockade by TMB.DesignFCs from healthy and GD patients were treated with combinations of TSH, M22, MG132 and AKTi (inhibitors of NF-?B and Akt, respectively), and TMB. TNF? protein production was measured by Luminex and flow cytometry. Messenger RNA expression was quantified by real time PCR.ResultsTreatment with TSH/M22 induced TNF? protein and mRNA production by FCs, both of which were reduced when FCs were pretreated with MG132 and AKTi (p<0.0001). TMB decreased TSH-induced TNF? protein production in circulating FCs from mean fluorescent index (MFI) value of 2.92 to 1.91, and mRNA expression in cultured FCs from 141- to 52-fold expression (p<0.0001). TMB also decreased M22-induced TNF? protein production from MFI of 1.67 to 1.12, and mRNA expression from 6- to 3-fold expression (p<0.0001).ConclusionTSH/M22 stimulates FC production of TNF? mRNA and protein. This process involves the transcription factor NF-?B and its regulator Akt. Blocking IGF-1R attenuates TSH/M22-induced TNF? production. This further delineates the interaction of TSHR and IGF1-R signaling pathways. By modulating the proinflammatory properties of FCs such as TNF? production, TMB may be a promising therapeutic agent for GO.

Project description:The molecular basis for anatomically dispersed clinical manifestations in Graves' disease (GD) eludes our understanding. Bone marrow-derived, pluripotent fibrocytes represent a subset of peripheral blood mononuclear cells and infiltrate the orbital and thyroid tissues in GD. These cells may be involved in the pathogenesis of thyroid-associated ophthalmopathy (TAO).The objective of the study was to quantify fibrocyte display of functional cell surface TSH receptor (TSHR), identify the profile of chemokines they express after TSHR activation, and determine whether circulating TSHR(+) peripheral blood fibrocytes are more frequent in situ in patients with TAO.Using a newly developed technique, fibrocytes were directly identified in peripheral blood from 31 patients with TAO and 19 healthy subjects receiving care at a multidisciplinary academic center.The frequency in situ of fibrocytes (collagen 1(+), CD45(+), CD34(+), CD14(+), CD86(+) peripheral blood mononuclear cells) was assessed by multiparameter flow cytometry and correlated to clinical disease activity and smoking status. Levels of TSHR-displaying fibrocytes and their response to TSH and TSHR-activating antibody, M22, were measured by flow cytometry, Luminex, and real-time PCR.The levels of TSHR expression by fibrocytes are substantially higher than those found in orbital fibroblasts. Moreover, the frequency of TSHR(+) fibrocytes in patients with TAO was greater than that in healthy subjects in situ. Their abundance is not influenced by disease activity or smoking history. These cells produce high levels of several cytokines and chemokines including IL-8, regulated upon activation, normal T cell expressed and secreted, and monocyte chemoattractant protein-1 when treated with TSH or M22. TSH induces IL-8 production at the pretranslational level. This induced cytokine can be detected in intact fibrocytes ex vivo.Frequency of circulating TSHR(+) fibrocytes is markedly increased in patients with TAO, and they express proinflammatory chemokines in response to TSH. Because they infiltrate both orbit and thyroid in GD, they may represent the link between systemic immunoreactivity and organ-specific autoimmunity.

Dataset Information

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

Publications

TSH Receptor Homodimerization in Regulation of cAMP Production in Human Thyrocytes <i>in vitro</i>.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets