Neutral Antibodies to the TSH Receptor Are Present in Graves’ Disease and Regulate Selective Signaling Cascades

Abstract

TSH receptor (TSHR) antibodies (Abs) may be stimulating, blocking, or neutral in their functional influences and are found in patients with autoimmune thyroid disease, especially Graves' disease (GD). Stimulators are known to activate the thyroid epithelial cells via both Gs- and Gq-coupled signaling pathways, whereas blockers inhibit the action of TSH and may act as weak agonists. However, TSHR neutral Abs do not block TSH binding and are unable to induce cAMP via Gsα. The importance of such neutral Abs in GD remains unclear because their functional consequence has been assumed to be zero. We hypothesized that: 1) neutral TSHR Abs are more common to GD than generally recognized; 2) they may induce distinct signaling imprints at the TSHR not seen with TSH itself; and 3) these signaling events may alter cellular function. To evaluate these hypotheses, we first confirmed the presence of neutral TSHR Abs in sera from patients with GD and then, using mouse and hamster neutral TSHR monoclonal Abs (N-mAbs) performed detailed signaling studies, including a proteomic Ab array, with rat thyrocytes (FRTL-5) as targets. This allowed us to examine a battery of signaling cascades and their downstream effectors. Neutral TSHR Abs were indeed frequently present in sera from patients with GD. Sixteen of 27 patients (59%) had detectable neutral TSHR Abs by competition assay with N-mAbs. On examining signaling cascades, we found that N-mAbs induced signal transduction, primarily via the protein kinase A II cascade. In addition to the activation of phosphatidylinositol 3K/Akt, N-mAbs, unlike TSH, had the ability to exclusively activate the mammalian target of rapamycin/p70 S6K, nuclear factor-κB, and MAPK-ERK1/2/p38α signaling cascades and their downstream effectors p90 ribosomal kinase/MAPK-interacting kinase-1/mitogen and stress-activated kinase-1 and N-mAbs activated all forms of protein kinase C isozymes. To define the downstream effector mechanisms produced by these signaling cascades, cytokine production, proliferation, and apoptosis in thyrocytes were investigated. Although N-mAbs produced less cytokines and proliferation compared with TSH, they had the distinction of inducing thyroid cell apoptosis under the experimental conditions used. When dissecting out possible mechanisms of apoptosis, we found that activation of multiple oxidative stress markers was the primary mechanism orchestrating the death signals. Therefore, using oxidative stress-induced apoptosis, N-mAbs may be capable of exacerbating the autoimmune response in GD via apoptotic cells inducing antigen-driven mechanisms. This may help explain the inflammatory nature of this common disorder.

Thyrotropin Receptor Stimulates Internalization-Independent Persistent Phosphoinositide Signaling

“…Thus, both C2 and M22 exhibit functionally selective short-term signaling (Strange, 2008). It had been shown previously that some thyroid-stimulating antibodies exhibit functionally selective signaling at TSHR (Morshed et al, 2009(Morshed et al, , 2010. It is noteworthy that there was a clear parallelism between the efficacies of these agonists to immediately and persistently stimulate cAMP and IP1 production.…”

Endogenous Allosteric Modulators of G Protein–Coupled Receptors

“…In Grave's disease, the autoimmune pathogenesis is well characterized and attributed to the ability of autoantibodies to bind the ectodomain of the TSHR, and either chronically activating it to cause hyperthyroidism (the most common form of Grave's disease; Orgiazzi et al, 1976) or inactivating it, causing hypothyroidism (Endo et al, 1978). Recently, neutral TSHR-directed antibodies have also been identified (Morshed et al, 2010). Some patients can swing between hyper-and hypothyroidism depending on the levels of stimulating or blocking TSHR autoantibodies (McLachlan and Rapoport, 2013).…”

Performance and Specificity of 6 Immunoassays for TSH Receptor Antibodies: A Multicenter Study

“…Antibodies (Ab) to the thyroid-stimulating hormone receptor (TSHR) may mimic [1][2][3] or block [4] the action of TSH or be functionally neutral [5]. TSHR stimulating Ab (TSAb) are responsible for many of the clinical manifestations of Graves' disease (GD) and are specific biomarkers [6][7][8][9][10] of this autoimmune thyroid disease (AITD).…”