Expression of endogenous putative TSH binding protein in orbit

Draman, Mohd Shazli, Grennan-Jones, Fiona, Taylor, Peter ORCID: https://orcid.org/0000-0002-3436-422X, Muller, Ilaria ORCID: https://orcid.org/0000-0003-2926-0722, Evans, Sam, Haridas, Anjana, Morris, Daniel S., Rees, Dafydd ORCID: https://orcid.org/0000-0002-1165-9092, Lane, Carol, Dayan, Colin ORCID: https://orcid.org/0000-0002-6557-3462, Zhang, Lei and Ludgate, Marian 2021. Expression of endogenous putative TSH binding protein in orbit. Current Issues in Molecular Biology 43 (3) , pp. 1794-1804. 10.3390/cimb43030126

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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.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	MRC Centre for Neuropsychiatric Genetics and Genomics (CNGG) Medicine
Additional Information:	This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).
Publisher:	MDPI
ISSN:	1467-3045
Date of First Compliant Deposit:	26 January 2022
Date of Acceptance:	20 October 2021
Last Modified:	10 Nov 2022 10:28
URI:	https://orca.cardiff.ac.uk/id/eprint/146952

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