Signal responses to neutral TSH receptor antibody – A cycle of damage in the pathophysiology of Graves’ disease

Graves’ disease is associated with TSH receptor (TSHR) antibodies of variable bioactivity including “neutral” antibodies (N-TSHR-Ab) that bind to the hinge region of the TSHR ectodomain. We have previously found that such antibodies induced thyroid cell apoptosis via excessive mitochondrial and ER stress with elevated reactive oxygen species (ROS). However, the detailed mechanisms by which excess ROS was induced remained unclear. To determine how ROS is induced by N-TSHR-monoclonal antibodies (mAb, MC1) mediated signaling and to measure stress in polyorganelles. Total ROS and mitochondrial ROS was measured by fluorometry of live rat thyrocytes. Live-cell imaging of labelled organelles was carried out using red or green fluorescent dyes. Proteins were detected by Li-Cor Western immunoblots and immunocytochemistry. Endocytosis of N-TSHR-mAb induced ROS, disturbed vesicular trafficking, damaged organelles and failed to induce lysosomal degradation and autophagy. We found that the endocytosis triggered signaling cascades involving Gα13 and PKC-δ leading to intrinsic thyroid cell apoptosis. These studies define the mechanism of ROS induction in thyroid cells following the endocytosis of N-TSHR-Ab/TSHR complexes. We suggest that a viscous cycle of stress initiated by cellular ROS and induced by N-TSHR-mAbs may orchestrate overt intra-thyroidal, retro-orbital, and intra-dermal inflammatory autoimmune reactions in patients with Graves’ disease.