Syed A. Morshed

The Thyroid-Stimulating Hormone Receptor: Impact of Thyroid-Stimulating Hormone and Thyroid-Stimulating Hormone Receptor Antibodies on Multimerization, Cleavage, and Signaling

The thyroid-stimulating hormone receptor (TSHR) has a central role in thyrocyte function and is also one of the major autoantigens for the autoimmune thyroid diseases. We review the post-translational processing, multimerization, and intramolecular cleavage of TSHR, all of which may modulate its signal transduction. The recent characterization of monoclonal antibodies to the TSHR, including stimulating, blocking, and neutral antibodies, have also revealed unique biologic insights into receptor activation and the variety of these TSHR antibodies may help explain the multiple clinical phenotypes seen in autoimmune thyroid diseases. Knowledge of the structure/function relationship of the TSHR is beginning to provide a greater understanding of thyroid physiology and thyroid autoimmunity.

Delineating the autoimmune mechanisms in Graves' disease.

The immunologic processes involved in autoimmune thyroid disease (AITD), particularly Graves’ disease (GD), are similar to other autoimmune diseases with the emphasis on the antibodies as the most unique aspect. These characteristics include a lymphocytic infiltrate at the target organs, the presence of antigen-reactive T and B cells and antibodies, and the establishment of animal models of GD by antibody transfer or immunization with antigen. Similar to other autoimmune diseases, risk factors for GD include the presence of multiple susceptibility genes, including certain HLA alleles, and the TSHR gene itself. In addition, a variety of known risk factors and precipitators have been characterized including the influence of sex and sex hormones, pregnancy, stress, infection, iodine and other potential environmental factors. The pathogenesis of GD is likely the result of a breakdown in the tolerance mechanisms, both at central and peripheral levels. Different subsets of T and B cells together with their regulatory populations play important roles in the propagation and maintenance of the disease process. Understanding different mechanistic in the complex system biology interplay will help to identify unique factors contributing to the AITD pathogenesis.

Stemness in Human Thyroid Cancers and Derived Cell Lines: The Role of Asymmetrically Dividing Cancer Stem Cells Resistant to Chemotherapy

CONTEXT Cancer stem cells (CSCs) have the ability to self-renew through symmetric and asymmetric cell division. CSCs may arise from mutations within an embryonic stem cell/progenitor cell population or via epithelial-mesenchymal transition (EMT), and recent advances in the study of thyroid stem cells have led to a growing recognition of the likely central importance of CSCs in thyroid tumorigenesis. OBJECTIVE The objectives of this study were to establish the presence of a stem cell population in human thyroid tumors and to identify, isolate, and characterize CSCs in thyroid cancer cell lines. RESULTS 1) Human thyroid cancers (n = 10) and thyroid cancer cell lines (n = 6) contained a stem cell population as evidenced by pluripotent stem cell gene expression. 2) Pulse-chase experiments with thyroid cancer cells identified a label-retaining cell population, a primary characteristic of CSCs, which at mitosis divided their DNA both symmetrically and asymmetrically and included a population of cells expressing the progenitor marker, stage-specific embryonic antigen 1 (SSEA-1). 3) Cells positive for SSEA-1 expressed additional stem cell markers including Oct4, Sox2, and Nanog were confirmed as CSCs by their tumor-initiating properties in vivo, their resistance to chemotherapy, and their multipotent capability. 4) SSEA-1-positive cells showed enhanced vimentin expression and decreased E-cadherin expression, indicating their likely derivation via EMT. CONCLUSIONS Cellular diversity in thyroid cancer occurs through both symmetric and asymmetric cell division, and SSEA-1-positive cells are one form of CSCs that appear to have arisen via EMT and may be the source of malignant thyroid tumor formation. This would suggest that thyroid cancer CSCs were the result of thyroid cancer transformation rather than the source.

Thyroid and Bone: Macrophage-Derived TSH-β Splice Variant Increases Murine Osteoblastogenesis

It is now firmly established that TSH may influence the physiology and patho-physiology of bone by activating osteoblasts and inhibiting osteoclast activity resulting in relative osteoprotection. Whether this influence is directly exerted by pituitary-derived TSH in vivo is less certain, because we have previously reported that the suppression of pituitary TSH does not remove such protection. Here, we have characterized the functional relevance of a novel form of the TSH-β subunit, designated TSH-βv, known to be produced by murine bone marrow cells. We found that fresh bone marrow-derived macrophages (MØs) preferentially produced TSH-βv and, when cocultured with CHO cells engineered to overexpress the full-length TSH receptor, were able to generate the production of intracellular cAMP; a phenomenon not seen in control CHO cells, such results confirmed the bioactivity of the TSH variant. Furthermore, cocultures of MØs and osteoblasts were shown to enhance osteoblastogenesis, and this phenomenon was markedly reduced by antibody to TSH-β, suggesting direct interaction between MØs and osteoblasts as observed under the electron microscope. These data suggest a new paradigm of local modulation of bone biology by a MØ-derived TSH-like molecule and raise the question of the relative contribution of local vs pituitary-derived TSH in osteoprotection.

A tyrosine residue on the TSH receptor stabilizes multimer formation.

Background The thyrotropin stimulating hormone receptor (TSHR) is a G protein coupled receptor (GPCR) with a large ectodomain. The ligand, TSH, acting via this receptor regulates thyroid growth and thyroid hormone production and secretion. The TSH receptor (TSHR) undergoes complex post –translational modifications including intramolecular cleavage and receptor multimerization. Since monomeric and multimeric receptors coexist in cells, understanding the functional role of just the TSHR multimers is difficult. Therefore, to help understand the physiological significance of receptor multimerization, it will be necessary to abrogate multimer formation, which requires identifying the ectodomain and endodomain interaction sites on the TSHR. Here, we have examined the contribution of the ectodomain to constitutive multimerization of the TSHR and determined the possible residue(s) that may be involved in this interaction. Methodology/Principal Findings We studied ectodomain multimer formation by expressing the extracellular domain of the TSHR linked to a glycophosphotidyl (GPI) anchor in both stable and transient expression systems. Using co-immunoprecipitation and FRET of tagged receptors, we established that the TSH receptor ectodomain was capable of multimerization even when totally devoid of the transmembrane domain. Further, we studied the effect of two residues that likely made critical contact points in this interaction. We showed that a conserved tyrosine residue (Y116) on the convex surface of the LRR3 was a critical residue in ectodomain multimer formation since mutation of this residue to serine totally abrogated ectodomain multimers. This abrogation was not seen with the mutation of cysteine 176 on the inner side of the LRR5, demonstrating that inter-receptor disulfide bonding was not involved in ectodomain multimer formation. Additionally, the Y116 mutation in the intact wild type receptor enhanced receptor degradation. Conclusions/Significance These data establish the TSH receptor ectodomain as one site of multimerization, independent of the transmembrane region, and that this interaction was primarily via a conserved tyrosine residue in LRR3.

T3 Regulates a Human Macrophage-Derived TSH-β Splice Variant: Implications for Human Bone Biology

TSH and thyroid hormones (T3 and T4) are intimately involved in bone biology. We have previously reported the presence of a murine TSH-β splice variant (TSH-βv) expressed specifically in bone marrow-derived macrophages and that exerted an osteoprotective effect by inducing osteoblastogenesis. To extend this observation and its relevance to human bone biology, we set out to identify and characterize a TSH-β variant in human macrophages. Real-time PCR analyses using human TSH-β-specific primers identified a 364-bp product in macrophages, bone marrow, and peripheral blood mononuclear cells that was sequence verified and was homologous to a human TSH-βv previously reported. We then examined TSH-βv regulation using the THP-1 human monocyte cell line matured into macrophages. After 4 days, 46.1% of the THP-1 cells expressed the macrophage markers CD-14 and macrophage colony-stimulating factor and exhibited typical morphological characteristics of macrophages. Real-time PCR analyses of these cells treated in a dose-dependent manner with T3 showed a 14-fold induction of human TSH-βv mRNA and variant protein. Furthermore, these human TSH-βv-positive cells, induced by T3 exposure, had categorized into both M1 and M2 macrophage phenotypes as evidenced by the expression of macrophage colony-stimulating factor for M1 and CCL-22 for M2. These data indicate that in hyperthyroidism, bone marrow resident macrophages have the potential to exert enhanced osteoprotective effects by oversecreting human TSH-βv, which may exert its local osteoprotective role via osteoblast and osteoclast TSH receptors.

Thyroid cell differentiation from murine induced pluripotent stem cells.

Background Here, we demonstrate the successful differentiation of induced pluripotent stem (iPS) cells into functional thyroid cells indicating the therapeutic potential of this approach when applied to individuals with thyroid deficiency. Research design and methods Using embryonic murine fibroblasts, we generated iPS cells with a single lentiviral “stem cell cassette” vector and then differentiated these iPS cells into thyroid cells after transfection with PAX8 and NKX2-1 by Activin A and TSH stimulation. Results The generated iPS cells expressed pluripotent stem cell markers as assessed using both reverse transcription quantitative PCRs and immunofluorescence staining with ~0.5% reprograming efficiency. Compared to control cells, the expression of thyroid-specific genes NIS, TSHR, Tg, and TPO were greatly enhanced in PAX8+NKX2-1+ iPS cells after differentiation. On stimulation with TSH, these differentiated iPS cells were also capable of dose-dependent cAMP generation and radioiodine uptake indicative of functional thyroid epithelial cells. Furthermore, the cells formed three-dimensional follicles in culture, and “thyroid organoids” formed after PAX8+NKX2-1+ iPS cells transplanted into nude mice, and all expressed Tg protein as judged immunohistochemically. Taken together, thyroid epithelial cells differentiated from iPS cells, which were themselves derived from murine fibroblasts, exhibited very similar properties to thyroid cells previously developed from traditional murine embryonic stem cells. Conclusion Thyroid cells differentiated from iPS cells offer the opportunity to examine the detailed transcriptional regulation of thyroid cell differentiation and may provide a useful future source for individualized regenerative cell therapy.

Stemness is Derived from Thyroid Cancer Cells

Background: One hypothesis for thyroid cancer development is its derivation from thyroid cancer stem cells (CSCs). Such cells could arise via different paths including from mutated resident stem cells within the thyroid gland or via epithelial to mesenchymal transition (EMT) from malignant cells since EMT is known to confer stem-like characteristics. Furthermore, EMT is a critical process for epithelial tumor progression, local invasion, and metastasis formation. In addition, stemness provides cells with therapeutic resistance and is the likely cause of tumor recurrence. However, the relevance of EMT and stemness in thyroid cancer progression has not been extensively studied. Methods: To examine the status of stemness in thyroid papillary cancer, we employed a murine model of thyroid papillary carcinoma and examined the expression of stemness and EMT using qPCR and histochemistry in mice with a thyroid-specific knock-in of oncogenic Braf (LSL-Braf(V600E)/TPO-Cre). This construct is only activated at the time of thyroid peroxidase (TPO) expression in differentiating thyroid cells and cannot be activated by undifferentiated stem cells, which do not express TPO. Results: There was decreased expression of thyroid-specific genes such as Tg and NIS and increased expression of stemness markers, such as Oct4, Rex1, CD15, and Sox2 in the thyroid carcinoma tissue from 6-week-old BRAFV600E mice indicating the dedifferentiated status of the cells and the fact that stemness was derived in this model from differentiated thyroid cells. The decreased expression of the epithelial marker E-cadherin and increased EMT regulators including Snail, Slug, and TGF-β1 and TGF-β3, and the mesenchymal marker vimentin demonstrated the simultaneous progression of EMT and the CSC-like phenotype. Stemness was also found in a cancer thyroid cell line (named Marca cells) derived from one of the murine tumors. In this cell line, we also found that overexpression of Snail caused up-regulation of vimentin expression and up-regulation of stemness markers Oct4, Rex1, and CD15, with enhanced migration ability of the cells. We also showed that TGF-β1 was able to induce Snail and vimentin expression in the Marca cell thyroid cancer line, indicating the induction of EMT in these cells, and this induction of EMT and stemness was significantly inhibited by celastro a natural inhibitor of neoplastic cells. Conclusion: Our findings support our earlier hypothesis that stemness in thyroid cancer is derived via EMT rather than from resident thyroid stem cells. In mice with a thyroid-specific knock-in of oncogenic Braf (LSL-Braf(V600E)/TPO-Cre), the neoplastic changes were dependent on thyroid cell differentiation and the onset of stemness must have been derived from differentiated thyroid epithelial cells. Furthermore, celastrol suppressed TGF-β1 induced EMT in thyroid cancer cells and may have therapeutic potential.

Antibody Protection Reveals Extended Epitopes on the Human TSH Receptor

Stimulating, and some blocking, antibodies to the TSH receptor (TSHR) have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD). However, successful crystallization of TSHR residues 22–260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs) have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1–412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity.

Biased signaling by thyroid-stimulating hormone receptor–specific antibodies determines thyrocyte survival in autoimmunity

Different autoantibodies against the thyroid-stimulating hormone receptor have distinct effects on receptor trafficking and thyrocyte survival. Antibodies determine thyrocyte fate Graves’ disease (GD) is an autoimmune disorder characterized by hyperthyroidism and the presence of autoantibodies against the thyroid-stimulating hormone receptor (TSHR), which are classified as stimulatory, blocking, or neutral and which bind to different receptor epitopes. Binding of TSH to the TSHR, a member of the GPCR superfamily, leads to the activation of G proteins and, in response to receptor phosphorylation, the activation of β-arrestin–dependent signaling. Through imaging analysis, Morshed et al. showed that exposure of thyrocytes to the stimulatory antibody S-TSHR-Ab led to G protein signaling, receptor internalization, and endosomal formation. In contrast, a neutral antibody (C-TSHR-Ab) induced β-arrestin–dependent signaling, but not G protein–dependent signaling, had defective trafficking, and accumulated intracellularly. This, in turn, triggered thyrocyte death by apoptosis. Together, these data suggest a mechanism by which different autoantibodies differentially affect thyrocyte homeostasis, which may have implications for the treatment of GD. The thyroid-stimulating hormone receptor (TSHR) is a heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptor (GPCR). Autoimmune hyperthyroidism, commonly known as Graves’ disease (GD), is caused by stimulating autoantibodies to the TSHR. We previously described TSHR-specific antibodies (TSHR-Abs) in GD that recognize linear epitopes in the cleavage region of the TSHR ectodomain (C-TSHR-Abs) and induce thyroid cell apoptosis instead of stimulating the TSHR. We found that C-TSHR-Abs entered the cell through clathrin-mediated endocytosis but did not trigger endosomal maturation and failed to undergo normal vesicular sorting and trafficking. We found that stimulating TSHR-Abs (S-TSHR-Abs) activated Gαs and, to a lesser extent, Gαq but that C-TSHR-Abs failed to activate any of the G proteins normally activated in response to TSH. Furthermore, specific inhibition of G proteins in the presence of S-TSHR-mAbs or TSH resulted in a similar failure of endosomal maturation as that caused by C-TSHR-mAbs. Hence, whereas S-TSHR-mAbs and TSH contributed to normal vesicular trafficking of TSHR through the activation of major G proteins, the C-TSHR-Abs resulted in GRK2- and β-arrestin-1–dependent biased signaling, which is interpreted as a danger signal by the cell. Our observations suggest that the binding of antibodies to different TSHR epitopes may decrease cell survival. Antibody-induced cell injury and the response to cell death amplify the loss of self-tolerance, which most likely helps to perpetuate GPCR-mediated autoimmunity.