Nuclear Targeting of IGF-1 Receptor in Orbital Fibroblasts from Graves' Disease: Apparent Role of ADAM17

Insulin-like growth factor-1 receptor (IGF-1R) comprises two subunits, including a ligand binding domain on extra- cellular IGF-1Rα and a tyrosine phosphorylation site located on IGF-1Rβ. IGF-1R is over-expressed by orbital fibroblasts in the autoimmune syndrome, Graves' disease (GD). When activated by IGF-1 or GD-derived IgG (GD-IgG), these fibroblasts produce RANTES and IL-16, while those from healthy donors do not. We now report that IGF-1 and GD-IgG provoke IGF-1R accumulation in the cell nucleus of GD fibroblasts where it co-localizes with chromatin. Nuclear IGF-1R is detected with anti-IGF-1Rα-specific mAb and migrates to approximately 110 kDa, consistent with its identity as an IGF-1R fragment. Nuclear IGF-1R migrating as a 200 kDa protein and consistent with an intact receptor was undetectable when probed with either anti-IGF-1Rα or anti-IGF-1Rβ mAbs. Nuclear redistribution of IGF-1R is absent in control orbital fibroblasts. In GD fibroblasts, it can be abolished by an IGF-1R-blocking mAb, 1H7 and by physiological concentrations of glucocorticoids. When cell-surface IGF-1R is cross-linked with 125I IGF-1, 125I-IGF-1/IGF-1R complexes accumulate in the nuclei of GD fibroblasts. This requires active ADAM17, a membrane associated metalloproteinase, and the phosphorylation of IGF-1R. In contrast, virally encoded IGF-1Rα/GFP fusion protein localizes equivalently in nuclei in both control and GD fibroblasts. This result suggests that generation of IGF-1R fragments may limit the accumulation of nuclear IGF-1R. We thus identify a heretofore-unrecognized behavior of IGF-1R that appears limited to GD-derived fibroblasts. Nuclear IGF-1R may play a role in disease pathogenesis

Increased Generation of Fibrocytes in Thyroid-Associated Ophthalmopathy

Context: The pathogenic basis for Graves’ disease (GD) continues to elude our understanding. Specifically why activating antibodies are generated against self-antigens remains uncertain as does the identity of the antigen(s) that provokes orbital involvement in GD, a process known as thyroid-associated ophthalmopathy (TAO)

IGF-1R protein differentially accumulates in the nuclei of TAO orbital fibroblasts and derives from the fibroblast surface.

<p>(<b>A</b>) Western blot analysis of nuclear and cytoplasmic IGF-1Rα in GD orbital fibroblasts before and following IGF-1 (10 nM) treatment for 16 h. Cells were subjected to subcellular fractionation as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034173#s4" target="_blank">Methods</a>” and membranes were probed with anti-IGF-1Rα, stripped, and re-probed with anti-Grb2 (cytoplasmic) and anti-c-Jun (nuclear) Abs. (<b>B</b>) Nuclear IGF-1Rα content in GD and control orbital fibroblasts before or following treatment with either IGF-1 (10 nM) or GD-IgG (15 µg/ml) for 16 hours. (<b>C</b>) Insulin fails to alter the nuclear content of IR or IGF-1Rα in GD orbital fibroblasts. Cells were treated with nothing or insulin (15 µg/ml) for 16 hrs. They were subjected to subcellular fractionation and Western blot analysis. (<b>D</b>) IGF-1Rβ (98 kDa) and the intact receptor (200 kDa) are undetectable in the nucleus under basal and IGF-1-treated conditions. (<b>E</b>) Control and GD fibroblasts were subjected to ¹²⁵I-IGF-1 cross-linking with either the cell-impermeable agent, BS, or the cell permeable agent, DSS. They were then treated with IGF-1. Nuclei were separated as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034173#s4" target="_blank">Methods</a>” and subjected to quantification of radioactivity. Results are representative of three experiments performed.</p

siRNA directed against ADAM17 attenuates the nuclear accumulation of IGF-1Rα in GD orbital fibroblasts.

<p>Fibroblasts were transfected with control siRNA or that specifically targeting ADAM 17. Some were left untreated while others were treated with IGF-1. Cells were fixed and stained with anti-IGF-1Rα Ab/Orgeon Green anti-rabbit Ab. Quantification of nuclear intensity was conducted so that each column represents the mean of ten randomly chosen nuclei ± SD. *, p<0.002 vs control; **, p<0.01 vs IGF-1. n = 3 independent determinations. (Inset) Adam17 siRNA or control siRNA was transfected into GD fibroblasts, cells were lysed, and proteins subjected to Western blot analysis by probing with anti-ADAM 17 Ab, stripping the membrane and incubating with anti-β-actin.</p

Confocal imaging of IGF-1R reveals nuclear accumulation in TAO fibroblasts following treatment with either IGF-1 or GD-IgG.

<p>(<b>A</b>) untreated (control, upper panels) or IGF-1 (10 nM) (lower panels) for 16 h. reveals localization of IGF-1Rα (green) as indicated by the yellow overlay nuclei in GD fibroblasts. Chromatin was stained by PI (red). Control fibroblasts failed to respond. The images labeled IGF-1Rβ (green) demonstrate an absence of effects with IGF-1 on nuclear accumulation. Scale bar, 25 µm. (<b>B</b>) IGF-1Rα (green) co-localizes in the PI-stained nuclei (yellow overlay) following treatment with GD-IgG (15 µg/ml) in GD but not in control fibroblasts. (<b>C</b>) Dexamethasone (10 nM) attenuates the nuclear accumulation of IGF-1Rα (<b>D</b>) as does the IGF-1R-blocking mAb, 1H7 (5 µg/ml). These experiments have been performed 4 times.</p

Virally encoded IGF-1Rα-GFP fusion protein expression in GD and control orbital fibroblasts.

<p>(<b>A</b>) Expression of the adenovirus-encoded IGF-1Rα–GFP fusion protein can be detected by Western blot analysis probed by both anti-GFP and anti-IGF-1Rα. (<b>B</b>) IGF-1Rα-GFP fusion protein accumulates in the nuclei of both control and GD orbital fibroblasts, as assessed by Western blot analysis following subcellular fractionation of nuclear protein. IGF-1 treatment fails to alter this pattern. These studies are representative of 5 experiments involving expression of virally encoded IGF-1Rα.</p

ADAM 17 appears to play a critical role in nuclear accumulation of IGF-1Rα.

<p>(<b>A</b>) GD orbital fibroblasts express ADAM 17 protein as assessed by Western blot. (<b>B</b>). TAPI-1, a specific inhibitor of ADAM17 activity, blocks the nuclear accumulation of IGF-1Rα provoked by IGF-1 in GD orbital fibroblasts. (<b>C</b>) Quantification of nuclear grey color signal intensity. Each bar in the histogram represents the mean ± SD of ten nuclei randomly chosen for each treatment group. *, p<0.05 vs IGF-1; **, p<0.01 vs control, n = 3 independent determinations.</p

IGF-1Rβ phosphorylation may play an essential role in the nuclear translocation of IGF-1Rα in GD orbital fibroblasts.

<p>(<b>A</b>) NVP-AEW-541, a specific tyrosine kinase inhibitor blocks IGF-1R phosphorylation (<b>B</b>) NVP-AEW-541 blocks nuclear IGF-1Rα accumulation. GD orbital fibroblasts were treated as indicated and stained with anti-IGF-1Rα Ab (green) and counterstained with PI. (<b>C</b>) Western blot analysis of nuclear proteins from GD orbital fibroblasts treated with nothing, IGF-1, NVP-AEW-541, or the combination and probed with anti- IGF-1Rα and anti-c-jun Abs. The findings are representative of three experiments performed.</p