Evidence for an association between thyroid-stimulating hormone and insulin-like growth factor 1 receptors: a tale of two antigens implicated in Graves' disease

Abstract

Thyroid-stimulating hormone receptor (TSHR) plays a central role in regulating thyroid function and is targeted by IgGs in Graves' disease (GD-IgG). Whether TSHR is involved in the pathogenesis of thyroid-associated ophthalmopathy (TAO), the orbital manifestation of GD, remains uncertain. TSHR signaling overlaps with that of insulin-like grow factor 1 receptor (IGF-1R). GD-IgG can activate fibroblasts derived from donors with GD to synthesize T cell chemoattractants and hyaluronan, actions mediated through IGF-1R. In this study, we compare levels of IGF-1R and TSHR on the surfaces of TAO and control orbital fibroblasts and thyrocytes and explore the physical and functional relationship between the two receptors. TSHR levels are 11-fold higher on thyrocytes than on TAO or control fibroblasts. In contrast, IGF-1R levels are 3-fold higher on TAO vs control fibroblasts. In pull-down studies using fibroblasts, thyrocytes, and thyroid tissue, Abs directed specifically against either IGF-1Rbeta or TSHR bring both proteins out of solution. Moreover, IGF-1Rbeta and TSHR colocalize to the perinuclear and cytoplasmic compartments in fibroblasts and thyrocytes by confocal microscopy. Examination of orbital tissue from patients with TAO reveals similar colocalization to cell membranes. Treatment of primary thyrocytes with recombinant human TSH results in rapid ERK phosphorylation which can be blocked by an IGF-1R-blocking mAb. Our findings suggest that IGF-1R might mediate some TSH-provoked signaling. Furthermore, they indicate that TSHR levels on orbital fibroblasts are considerably lower than those on thyrocytes and that this receptor associates with IGF-1R in situ and together may comprise a functional complex in thyroid and orbital tissue.

Figure 1

Orbital fibroblasts from patients with TAO exhibit increased cell surface IGF-1R display but TSHR levels appear extremely low and similar to those in controls. Fibroblasts were stained with anti-IGF-1R or anti-TSHR Abs as described in “Methods” and subjected to flow cytometry. The grey open histograms (IGF-1R) and dotted histograms (TSHR) represent staining with isotype control Abs. The data represent results from 3 different donors with TAO and 3 without autoimmune disease.

Figure 2

(A) Multiple strains of orbital fibroblasts from patients with TAO (n=9) express increased IGF-1R expression compared to fibroblasts from control donors (n=5). IGF-1R levels were determined and expressed as a multiple of isotype fluorescence (Mean fluorescent Intensity Multiplier; *p<0.003). (B) IGF-1R molecule density is elevated in multiple strains of orbital fibroblasts from patients with TAO (n=5) compared to control strains (n=4). Quantification of the relative Ab binding to cell surface IGF-1R was determined by ABC analysis, as described in “Methods” section (*p<0.01 TAO vs control). (C) TSHR molecular densities are extremely low and similar in multiple strains of orbital fibroblasts from patients TAO GD (n=5) compared to control strains (n=5). Thyrocyte strains (n=3) display substantially higher levels of TSHR. Quantification of relative Ab binding to TSHR was determined by ABC analysis, as described in “Methods” section (*p< 10^-6, fibroblasts vs thyrocytes).

Figure 3

(A) Paired analysis of IGF-1R (left panels) and TSHR (right panels) in orbital fibroblasts from patients with TAO (top) and thyrocytes (bottom). Orbital fibroblasts and thyrocytes were stained with anti-IGF-1R or anti TSHR Abs as described in “Methods” and subjected to flow cytometry. Dotted histograms represent staining with isotype control Abs. (B) Orbital fibroblasts from control donors and those with TAO were subjected to standard incubation (-DM) or the medium described in “Methods” to promote differentiation into adipocytes (+DM). They were then stained with either anti-TSHR Abs or Oil Red O. The former were subjected to flow cytometry and the latter were examined by light microscopy (insets).

Figure 4

Effects of IGF-1 and rTSH on proliferation of orbital fibroblasts from donors without (control) or with Graves’ disease. IGF-1 enhances expansion of orbital fibroblasts from patients with TAO compared to control fibroblasts while TSH does not. Cell number was assessed as described in “Methods” after 7 days in culture with IGF-1 (10 or 50 ng/ml) or TSH (2 mU/ml). Data are expressed as the mean ± SEM derive from 5 independent experiments (* p<0.05 vs control).

Figure 5

(panels A-F). Immunofluorescence staining for IGF-1Rβ (red) and TSHR (green) demonstrates co-localization of the two proteins (yellow) by confocal microscopy in orbital fibroblasts from a patient with severe TAO (panels A-C) and thyrocytes (panels D-F). In the merged images, co-localization appears as yellow or orange (panels C and F). Panels G-I, Confocal images using a different pair of Abs show IGF-1R β (green) and TSHR (red), and co-localization of IGF-1Rβ and TSHR (yellow) in fibroblasts. Note a similar co-localization pattern of these receptors. Panels J-L, Immunofluorescence staining for TSHR (green) and IGF-1Rα (red) in orbital fibroblasts demonstrates a different co-localization pattern than that for IGF-1Rβ. The merged image (panel L) demonstrates co-localization (yellow to orange). Orbital fibroblasts and thyrocytes were cultured and fixed as described in “Methods” and visualized with a Nikon PCM 2000 confocal microscope. Scale bar, 25 μm. Orbital connective tissue from a patient with TAO also stains for TSHR (panel M, green), IGF-1R (panel N, red), and also demonstrated receptor co-localization when the images are merged (panel O, orange). In sets in Panels J and K, isotype control Abs.

Figure 6

(A and B) Western blot analysis of proteins from orbital fibroblasts, thyrocytes and thyroid tissue subjected to immunoprecipitation with either anti-IGF-1Rβ or anti-TSHR antibodies. Confluent cultures were harvested. Cells and tissue were solubilized and subjected to precipitation (IP) with anti-IGF-1Rβ, anti-TSHR or anti-IL-6 receptor Abs (control) as described in “Methods”. Precipitated proteins were separated with SDS-PAGE, transferred and immunoblotted with (IB) anti-IGF-1Rα, anti-IGF-1Rβ or anti-TSHR Abs. (C) Knocking-down IGF-1R expression with specific siRNA disrupts the TSHR/IGF-1Rβ complex. Cells were prepared as described above after they had been treated with siRNA for IGF-1R. They were then subjected to Western analysis (upper0 or IP (lower) and probed as indicated in the figure. (D) Western blot analysis of ERK activation in confluent thyrocytes treated with IGF-1 (10 ng/ml), rhTSH (1 mU/ml) or GD-IgG (100 ng/ml) without or with the blocking anti-IGF-1R mAb, 1H7 (5 μg/ml) for 15 min. Cells were harvested and proteins subjected to western blot analysis for phospho-ERK levels at 42/44 kDa. Loading equivalence was confirmed by blotting with anti-β actin. Signals were generated as described in “Methods”. Relative densities, corrected for their respective β-actin signals were: control, 0.250; IGF-1, 0.775; IGF-1 + 1H7, 0.1962; TSH, 0.432; TSH + 1H7, 0.053; GD-IgG, 0.506; GD-IgG + 1H7, 0.0439