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

Abstract

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. We further conclude that the preponderance of evidence supports TSHR as the direct and only target of stimulating autoantibodies in GO and maintain that the TSHR should remain a major target for further development of a medical therapy for GO in concert with drugs that target TSHR/IGF1R crosstalk.

Keywords: Graves’ orbital fibroblasts; Graves’ orbitopathy; IGF1R; IGF1R antibodies; TSHR; TSHR antibodies; hyaluronan; receptor crosstalk.

Figure 1

(A) model of thyroid-stimulating hormone receptor/insulin-like growth factor 1 receptor (TSHR/IGF1R) crosstalk initiated by M22 binding to TSHR on hyaluronan secretion by Graves’ orbital fibroblasts. Graves’ orbitopathy fibroblasts (GOFs) secrete hyaluronan following stimulation by M22, a human monoclonal TSHR-stimulating antibody. This response to increasing doses of M22 is biphasic (panel A), as shown by the light and dark gray regions of the curve. The light gray region is the IGF1R-dependent (high potency) phase, which was found by We to be inhibited by linsitinib, an IGF1R kinase inhibitor [27]. The dark gray region indicates the IGF1R-independent (low potency) phase of hyaluronan secretion, which occurs regardless of crosstalk with IGF1R. The cartoon in panel (B) depicts the two pathways that mediate TSHR-initiated hyaluronan secretion. The combination of these two pathways leads to full stimulation of hyaluronan. Adapted from [27].

Figure 2

M22 specificity to TSHR demonstrated by competitive binding assay. The human monoclonal antibody M22 was coupled to the fluorescent probe Alexa-647 (Alexa-647-M22), and its binding to cell surface receptors was measured by flow cytometry. Binding of M22-647 was conducted in the presence of excess amounts of unlabeled TSHR agonists (M22, KSAb1, and TSH) and anti-IGF1R antibodies (AF305 and 1H7). Parental HEK293 (HEK-EM) cells do not express TSHRs but endogenously express IGF1Rs, and HEK-TSHR cells overexpress TSHRs and endogenously express IGF1Rs. Gray histograms (all panels) illustrate nonspecific cell autofluorescence. Black histograms (panels C–H) illustrate specific Alexa-647-M22 binding. Panels A and B: In HEK-EM cells, there is no specific binding. Panel C: specific binding that is totally inhibited (panel D) by an excess of unlabeled M22. Panel E: another unlabeled mouse monoclonal TSHR antibody, KSAb1, completely inhibits specific binding. Panel F: unlabeled TSH partially inhibits specific binding. Panels G and H: unlabeled anti-IGF1R blocking antibodies, AF305 and 1H7, have no effect on specific Alexa-647-M22 binding. Reprinted from [8].

Figure 3

Anti-IGF1R blocking antibodies may or may not inhibit GO-Ig stimulation of hyaluronan secretion by Graves’ orbital fibroblasts. GOFs were stimulated with purified GO-Igs in the presence of IGF1R blocking antibodies, AF305 (white circles) and 1H7 (gray squares). Data were normalized to their own control (dotted line). Each data point represents GO-Igs from one patient, and black bars indicate the averaged means of all stimulatory GO-Igs. Both AF305 and 1H7 completely inhibit binding of IGF1 to IGF1Rs [11]. While AF305 had no effect on GO-Ig stimulation of hyaluronan (HA) secretion, 1H7 partially inhibited GO-Ig stimulation of HA secretion. These findings are consistent with the idea that AF305 does not affect TSHR/IGF1R crosstalk, whereas 1H7 inhibits crosstalk. Reprinted from [11]. **** P < 0.001 vs. GO-Ig control by Student’s t-test.

Figure 4

IGF1Rs are not activated by GO-Igs in Graves’ orbital fibroblasts. IGF1, the human monoclonal antibody M22 and 57 GO-Ig preparations were used to determine whether GO-Igs stimulated the phosphorylation of IGF1R in GOFs. Rapid autophosphorylation of IGF1R is the main initiator of IGF1R signaling. As expected, IGF1 stimulated a robust increase in phospho-IGF1R, but M22 had no effect. None of the 57 GO-Ig preparations tested increased phospho-IGF1R. Reprinted from [11].

Figure 5

Model of the effects of IGF1R and TSHR antagonists on GO- immunoglobulin (Ig) stimulation of hyaluronan secretion by Graves’ orbital fibroblasts. GO-Igs bind directly to and activate TSHR, stimulating two signaling pathways: IGF1R-independent (dark gray arrow) and IGF1R-dependent (light gray arrows), leading to secretion of hyaluronan. TSHR/IGF1R crosstalk is initiated by binding of GO-Igs to TSHR and requires TSHR and IGF1R to be in close proximity within a signalosome. Synergistic interactions between TSHR and IGF1R occur rapidly at phosphorylation of ERK, demonstrating that crosstalk between TSHR and IGF1R occurs early in the signaling cascade, proximal to the receptors [30]. β-arrestin 1 (βARR) is essential for GO-Ig stimulation of ERK phosphorylation, and hyaluronan secretion and proximity ligation assays in GOFs demonstrated that β-arrestin 1 physically scaffolds TSHRs and IGF1Rs in a protein complex [35]. These pathways appear not to involve cAMP, which has been considered a canonical pathway of TSHR in thyrocytes. However, recent evidence has demonstrated β-arrestin-mediated signaling in thyrocytes and osteoblast-like cells also. Some inhibitory IGF1R antibodies (IGF1RAb), such as 1H7 and probably teprotumumab, bind to IGF1Rs and inhibit activation of IGF1R by activated TSHR via crosstalk. TSHR antagonists, including drug-like small molecule inhibitors, anti-TSHR antibodies and cyclic peptides via immune hyposensitization inhibit signaling initiated by GO-Ig activation of TSHR, and thereby totally abolish both pathways of stimulation of hyaluronan secretion. Combination therapy with TSHR and IGF1R antagonists could minimize drug side effects and, therefore, may have therapeutic benefits [46].