Expression of Endogenous Putative TSH Binding Protein in Orbit

Area of Science:

• Endocrinology
• Immunology
• Cell Biology

Background:

• Graves' disease is an autoimmune disorder caused by thyroid stimulating antibodies (TSAB).
• Graves' Orbitopathy (GO) is an associated autoimmune complication affecting orbital tissues.
• The role of TSH binding proteins, specifically truncated TSHR variants (TSHRv), and nonclassical ligands like thyrostimulin (α2β5) in GO pathogenesis is not fully understood.

Purpose of the Study:

• To investigate the expression and function of truncated TSHR variants (TSHRv) in human orbital preadipocyte-fibroblasts (OF) from GO patients and non-GO individuals.
• To determine if TSHRv presence or levels correlate with GO.
• To explore the potential of TSHRv to modulate TSHR signaling and TSAB activity.

Main Methods:

• Primary human orbital preadipocyte-fibroblasts (OF) from GO and non-GO subjects were cultured and differentiated.
• Quantitative PCR (QPCR) and Western blot were used to measure TSHRv transcript and protein expression.
• Cyclic-AMP production was assessed via luciferase-reporter and RIA assays upon stimulation with TSH or monoclonal TSAB (M22).
• Bioassays were conducted using culture media (CM) from differentiated OF to evaluate TSHRv neutralizing capacity.

Main Results:

• TSHRv transcript and protein levels were significantly higher in OF from GO patients compared to non-GO individuals (p = 0.039).
• TSHRv protein was detected in both cell lysates and culture media.
• Cyclic-AMP response to TSH or M22 stimulation was modulated by differentiation and inversely correlated with the TSHRv:TSHR ratio.
• Culture media from differentiated GO-OF showed reduced TSH/M22-induced signaling, suggesting secreted TSHRv neutralized antibody effects.
• α2 transcripts were present but decreased during adipogenesis; β5 transcripts were barely detectable.

Conclusions:

• Truncated TSHR variants (TSHRv) are expressed as proteins in human orbital fibroblasts and are more abundant in GO patients.
• TSHRv can be secreted and appear to neutralize the effects of TSH and TSABs, suggesting a regulatory role in TSHR signaling.
• These findings indicate that TSHRv may play a significant role in the pathogenesis or modulation of Graves' Orbitopathy.