Expression of Endogenous Putative TSH Binding Protein in Orbit.
Mohd Shazli DramanFiona Grennan-JonesPeter TaylorIlaria MullerSam EvansAnjana HaridasDaniel S MorrisD Aled ReesCarol LaneColin DayanLei ZhangMarian LudgatePublished in: Current issues in molecular biology (2021)
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.
Keyphrases
- binding protein
- poor prognosis
- end stage renal disease
- endothelial cells
- protein kinase
- single cell
- ejection fraction
- induced apoptosis
- south africa
- newly diagnosed
- peritoneal dialysis
- adipose tissue
- high throughput
- crispr cas
- chronic kidney disease
- protein protein
- cell therapy
- gene expression
- oxidative stress
- small molecule
- diabetic rats
- stem cells
- patient reported outcomes
- copy number
- amino acid
- dna methylation
- heat shock protein
- insulin resistance
- extracellular matrix
- mesenchymal stem cells
- cell proliferation
- patient reported
- quantum dots
- sensitive detection