Rauf Latif

Thyrotropin receptor–associated diseases: from adenomata to Graves disease

The thyroid-stimulating hormone receptor (TSHR) is a G protein-linked, 7-transmembrane domain (7-TMD) receptor that undergoes complex posttranslational processing unique to this glycoprotein receptor family. Due to its complex structure, TSHR appears to have unstable molecular integrity and a propensity toward over- or underactivity on the basis of point genetic mutations or antibody-induced structural changes. Hence, both germline and somatic mutations, commonly located in the transmembrane regions, may induce constitutive activation of the receptor, resulting in congenital hyperthyroidism or the development of actively secreting thyroid nodules. Similarly, mutations leading to structural alterations may induce constitutive inactivation and congenital hypothyroidism. The TSHR is also a primary antigen in autoimmune thyroid disease, and some TSHR antibodies may activate the receptor, while others inhibit its activation or have no influence on signal transduction at all, depending on how they influence the integrity of the structure. Clinical assays for such antibodies have improved significantly and are a useful addition to the investigative armamentarium. Furthermore, the relative instability of the receptor can result in shedding of the TSHR ectodomain, providing a source of antigen and activating the autoimmune response. However, it may also provide decoys for TSHR antibodies, thus influencing their biological action and clinical effects. This review discusses the role of the TSHR in the physiological and pathological stimulation of the thyroid.

The TSH receptor reveals itself

The thyrotropin receptor (TSHR), one of the primary antigens in autoimmune thyroid disease, is a target of both antigen-specific T cells and antibodies in patients with this condition (1). Autoantibodies to the TSHR (TSHR-Ab) act as thyroid stimulating factor (TSH) agonists in autoimmune hyperthyroidism (Robert Graves disease) but as TSH antagonists in autoimmune hypothyroidism (Hashimoto thyroiditis). The TSHR antigen is primarily expressed in the epithelial cells of the thyroid follicles, but TSHR mRNA and protein have been reported in a variety of cell types, some of which show evidence of receptor activity (Table ​(Table11). Table 1 Tissue distribution of the TSHR The TSHR gene, cloned in 1989 (2–5), maps to human chromosome 14q and encodes a predicted seven-transmembrane, G protein–coupled glycoprotein. Although it is similar to the luteinizing hormone receptor and the follicle-stimulating hormone receptor, the TSHR is the largest of the glycoprotein hormone receptors, due primarily to 8– and 50–amino acid insertions in its ectodomain (residues 38–45 and 317–367) (6). As predicted from its cDNA, the TSHR has an unglycosylated molecular weight of 84 kDa but the glycosylated holoreceptor runs on SDS-PAGE with an apparent molecular weight of 95–100 kDa. There are six potential N-linked glycosylation sites on the TSHR, and it was recently shown that the TSHR is also palmitoylated (7). The minimal 5′ promoter region required to confer thyroid-specific expression and cAMP autoregulation extends from 220 to 39 bp upstream of the transcription start site, but there are multiple transcription start sites between –89 and –68 bp (8).

A monoclonal thyroid-stimulating antibody

The thyrotropin receptor, also known as the thyroid-stimulating hormone receptor (TSHR), is the primary antigen of Graves disease. Stimulating TSHR antibodies are the cause of thyroid overstimulation and were originally called long-acting thyroid stimulators due to their prolonged action. Here we report the successful cloning and characterization of a monoclonal antibody (MS-1) with TSHR-stimulating activity. The thyroid-stimulating activity of MS-1 was evident at IgG concentrations as low as 20 ng/ml. MS-1 also competed for radiolabeled TSH binding to the native TSHR and was able to compete for TSH-induced stimulation. MS-1 recognized a conformational epitope within the TSHR alpha (or A) subunit but excluding the receptor cleavage region. Using an assay measuring loss of antibody recognition after cleavage we demonstrated that MS-1, in contrast to TSH, was unable to enhance TSHR posttranslational cleavage. Since receptor cleavage is followed by alpha subunit shedding and receptor degradation, the functional half-life of the receptor may be extended. The isolation and characterization of MS-1 provides a novel explanation for the prolonged thyroid stimulation in this disease which may be secondary to the lack of receptor cleavage in addition to the prolonged half-life of IgG itself.

Intermittent recombinant TSH injections prevent ovariectomy-induced bone loss

We recently described the direct effects of thyroid-stimulating hormone (TSH) on bone and suggested that the bone loss in hyperthyroidism, hitherto attributed solely to elevated thyroid hormone levels, could at least in part arise from accompanying decrements in serum TSH. Recent studies on both mice and human subjects provide compelling evidence that thyroid hormones and TSH have the opposite effects on the skeleton. Here, we show that TSH, when injected intermittently into rodents, even at intervals of 2 weeks, displays a powerful antiresorptive action in vivo. By virtue of this action, together with the possible anabolic effects shown earlier, TSH both prevents bone loss and restores the lost bone after ovariectomy. Importantly, the osteoclast inhibitory action of TSH persists ex vivo even after therapy is stopped for 4 weeks. This profound and lasting antiresorptive action of TSH is mimicked in cells that genetically overexpress the constitutively active ligand-independent TSH receptor (TSHR). In contrast, loss of function of a mutant TSHR (Pro → Leu at 556) in congenital hypothyroid mice activates osteoclast differentiation, confirming once again our premise that TSHRs have a critical role in regulating bone remodeling.

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.

Hyperthyroid-associated osteoporosis is exacerbated by the loss of TSH signaling.

The osteoporosis associated with human hyperthyroidism has traditionally been attributed to elevated thyroid hormone levels. There is evidence, however, that thyroid-stimulating hormone (TSH), which is low in most hyperthyroid states, directly affects the skeleton. Importantly, Tshr-knockout mice are osteopenic. In order to determine whether low TSH levels contribute to bone loss in hyperthyroidism, we compared the skeletal phenotypes of wild-type and Tshr-knockout mice that were rendered hyperthyroid. We found that hyperthyroid mice lacking TSHR had greater bone loss and resorption than hyperthyroid wild-type mice, thereby demonstrating that the absence of TSH signaling contributes to bone loss. Further, we identified a TSH-like factor that may confer osteoprotection. These studies suggest that therapeutic suppression of TSH to very low levels may contribute to bone loss in people.

Blocking antibody to the β-subunit of FSH prevents bone loss by inhibiting bone resorption and stimulating bone synthesis

Low estrogen levels undoubtedly underlie menopausal bone thinning. However, rapid and profuse bone loss begins 3 y before the last menstrual period, when serum estrogen is relatively normal. We have shown that the pituitary hormone FSH, the levels of which are high during late perimenopause, directly stimulates bone resorption by osteoclasts. Here, we generated and characterized a polyclonal antibody to a 13-amino-acid-long peptide sequence within the receptor-binding domain of the FSH β-subunit. We show that the FSH antibody binds FSH specifically and blocks its action on osteoclast formation in vitro. When injected into ovariectomized mice, the FSH antibody attenuates bone loss significantly not only by inhibiting bone resorption, but also by stimulating bone formation, a yet uncharacterized action of FSH that we report herein. Mesenchymal cells isolated from mice treated with the FSH antibody show greater osteoblast precursor colony counts, similarly to mesenchymal cells isolated from FSH receptor (FSHR)−/− mice. This suggests that FSH negatively regulates osteoblast number. We confirm that this action is mediated by signaling-efficient FSHRs present on mesenchymal stem cells. Overall, the data prompt the future development of an FSH-blocking agent as a means of uncoupling bone formation and bone resorption to a therapeutic advantage in humans.

Blocking FSH induces thermogenic adipose tissue and reduces body fat

Menopause is associated with bone loss and enhanced visceral adiposity. A polyclonal antibody that targets the β-subunit of the pituitary hormone follicle-stimulating hormone (Fsh) increases bone mass in mice. Here, we report that this antibody sharply reduces adipose tissue in wild-type mice, phenocopying genetic haploinsufficiency for the Fsh receptor gene Fshr. The antibody also causes profound beiging, increases cellular mitochondrial density, activates brown adipose tissue and enhances thermogenesis. These actions result from the specific binding of the antibody to the β-subunit of Fsh to block its action. Our studies uncover opportunities for simultaneously treating obesity and osteoporosis.

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.

The Emerging Cell Biology of Thyroid Stem Cells

CONTEXT Stem cells are undifferentiated cells with the property of self-renewal and give rise to highly specialized cells under appropriate local conditions. The use of stem cells in regenerative medicine holds great promise for the treatment of many diseases, including those of the thyroid gland. EVIDENCE ACQUISITION This review focuses on the progress that has been made in thyroid stem cell research including an overview of cellular and molecular events (most of which were drawn from the period 1990-2011) and discusses the remaining problems encountered in their differentiation. EVIDENCE SYNTHESIS Protocols for the in vitro differentiation of embryonic stem cells, based on normal developmental processes, have generated thyroid-like cells but without full thyrocyte function. However, agents have been identified, including activin A, insulin, and IGF-I, which are able to stimulate the generation of thyroid-like cells in vitro. In addition, thyroid stem/progenitor cells have been identified within the normal thyroid gland and within thyroid cancers. CONCLUSIONS Advances in thyroid stem cell biology are providing not only insight into thyroid development but may offer therapeutic potential in thyroid cancer and future thyroid cell replacement therapy.