Background: TSH and IGF-1 signals have been shown to be additive in their action on thyroid cells and fibroblasts and this coordination emphasizes the role of TSH receptor (TSHR) and IGF-1 receptor (IGF-1R) signaling exerted by stimulating TSHR antibodies in patients with Thyroid Eye Disease (TED). Here we have reanalyzed the influence of TSHR autoantibodies on TSHR and IGF-1R expression in a fibroblast model.

Methods: We developed a new method to assess TSHR expression using a hinge-region monoclonal antibody (MC-1) that does not interfere with TSH or TSHR antibodies. Using molecular dynamic simulations we first assessed access to the binding sites for TSH and TSHR antibodies within the leucine rich repeat (LRR) region of the TSHR ectodomain in the presence of MC-1 binding. 3T3-L1 fibroblasts and human orbital fibroblasts were treated with increasing doses of TSH or the stimulating TSHR antibody M22. TSHR and IGF1R expressions were measured by flow cytometry and qPCR, with 3T3-L1 cell differentiation confirmed by Oil Red O staining. To explore mechanisms of TSH- and M22-induced receptor upregulation, we assessed the effects of inhibiting protein synthesis and endocytosis.

Results: TSH ligand induced a dose dependent increase in detectable expression of the TSHR as measured using MC-1 binding. Furthermore, parallel assessment of IGF-1R expression showed a similar dose dependent increase in expression induced by TSH. Studies with stimulating TSHR antibody (M22) also demonstrated a similar increase in TSHR and IGF-1R expression peaking at 48 hours. These data could not be explained by transcriptional changes as measured by qPCR. In addition, analysis of the effect of protein synthesis inhibition failed to reduce TSHR and IGF1R induction. However, inhibition of endocytosis markedly reduced enhancement of TSHR and IGF-1R expression.

Conclusions: These data strengthen the role of TSHR antibodies in TED by showing that they enhance TSHR and IGF-1R expression in fibroblasts, helping to explain their additive retroorbital signaling. This enhancement is largely driven by receptor recycling, which can be blocked by inhibiting endocytosis and may be therapeutically targeted. The dual increase in both receptors also supports prior evidence of TSHR/IGF-1R complex formation.