Expression of Endogenous Putative TSH Binding Protein in Orbit

Abstract

Thyroid stimulating antibodies (TSAB) cause Graves’ disease and contribute to Graves’ Orbitopathy (GO) pathogenesis. We hypothesise that the presence of TSH binding proteins (truncated TSHR variants (TSHRv)) and/or nonclassical ligands such as thyrostimulin (α2β5) might provide a mechanism to protect against or exacerbate GO. We analysed primary human orbital preadipocyte-fibroblasts (OF) from GO patients and people free of GO (non-GO). Transcript (QPCR) and protein (western blot) expression levels of TSHRv were measured through an adipogenesis differentiation process. Cyclic-AMP production by TSHR activation was studied using luciferase-reporter and RIA assays. After differentiation, TSHRv levels in OF from GO were significantly higher than non-GO (p = 0.039), and confirmed in ex vivo analysis of orbital adipose samples. TSHRv western blot revealed a positive signal at 46 kDa in cell lysates and culture media (CM) from non-GO and GO-OF. Cyclic-AMP decreased from basal levels when OF were stimulated with TSH or Monoclonal TSAB (M22) before differentiation protocol, but increased in differentiated cells, and was inversely correlated with the TSHRv:TSHR ratio (Spearman correlation: TSH r = −0.55, p = 0.23, M22 r = 0.87, p = 0.03). In the bioassay, TSH/M22 induced luciferase-light was lower in CM from differentiated GO-OF than non-GO, suggesting that secreted TSHRv had neutralised their effects. α2 transcripts were present but reduced during adipogenesis (p < 0.005) with no difference observed between non-GO and GO. β5 transcripts were at the limit of detection. Our work demonstrated that TSHRv transcripts are expressed as protein, are more abundant in GO than non-GO OF and have the capacity to regulate signalling via the TSHR.

A review of TSHR- and IGF-1R-related pathogenesis and treatment of Graves’ orbitopathy

“…The findings of Woeller et al (54) also demonstrate that TSHR signalling in GO-OFs stimulates proliferation indirectly through induction of miR-146a and miR-155, reducing the expression of ZNRF3 and PTEN that normally block cell proliferation. The latest interesting study by Draman MS et al suggests a variant in TSHR that might explain the antigenic role of this receptor in OF even prior to adipogenesis (55). In conclusion, TSHR plays a specific role in GO pathogenesis and has become a therapeutic target.…”

“…The latest interesting study by Draman MS et al. suggests a variant in TSHR that might explain the antigenic role of this receptor in OF even prior to adipogenesis ( 55 ). In conclusion, TSHR plays a specific role in GO pathogenesis and has become a therapeutic target.…”

Orbital Signaling in Graves’ Orbitopathy

“…Furthermore, induced murine models of GD and GO are more effective when immunizing with the A subunit than with the complete TSHR ( 74 , 75 ). We have reported that the 1.3 variant is expressed as a protein and can affect TSHR activation ( 76 ). Thus, these variants could have impact on the pathogenesis of GO by inducing further production of TSAB or protect against GO by ‘neutralizing’ TSAB, respectively.…”

ELF3-induced miR-182 inhibits adipogenic differentiation in Graves’ orbitopathy by targeting thyrotropin receptor

“…The thyrotropin receptor (TSHR) is a G protein coupled receptor found in thyroid follicular epithelial cells, adipocytes, osteocytes and cardiomyocytes [14]. It has been reported that TSHR was upregulated along with intraorbital preadipocytes of GO patients differentiated into mature adipocytes [15][16][17]. The PI3K/AKT pathway is a known TSHR signal transduction pathway in Graves' disease [18].…”