Evidence for protein and mRNA TSHr expression in fibroblasts from patients with thyroid-associated ophthalmopathy (TAO) after adipocytic differentiation.
OBJECTIVE
Thyroid-associated ophthalmopathy (TAO) is a chronic autoimmune disorder characterized by an increased volume of adipose/connective tissue in the human orbit.


METHODS
The aim of this study was to investigate the thyrotropin receptor (TSHr) expression in orbital fibroblasts from TAO patients undergoing adipocytic differentiation.


METHODS
Retro-ocular tissue and skin were obtained from five patients undergoing orbital decompression surgery for TAO and placed in culture. Proliferating fibroblasts were subjected to adipocytic differentiation for 10 days. Total RNA was isolated from fibroblasts and was reverse transcribed. TSHr mRNA levels were determined by real-time PCR. cAMP was determined by radioimmunoassay (RIA) after fibroblast incubation with the substances to test.


RESULTS
Orbital differentiated fibroblasts became rounded and acquired lipid droplets. The amount of TSHr mRNA in these fibroblasts was higher than fibroblasts not subjected to adipocytic differentiation. Immunocytochemical analysis showed TSHr protein in differentiated orbital fibroblasts. Differentiated orbital fibroblasts stimulated with bovine (b) TSH showed a cAMP production greater than that in paired undifferentiated cultures. A specific thyroid-inhibiting antibody (TBAb) inhibited cAMP production after bTSH challenge, and a thyroid-stimulating antibody (TSAb) stimulated cAMP production in differentiated fibroblasts.


CONCLUSIONS
We suggest that orbital fibroblasts subjected to adipocytic differentiation increase TSHr expression that responds specifically to bTSH and TSAb stimulation, and to TBAb inhibition.
Agretti
Patrizia
P
Dipartimento di Endocrinologia e Metabolismo, Centro Eccellenza AmbiSEN Università di Pisa, Via Paradisa 2, 56124 Pisa, Italy.
De Marco
Giuseppina
G
De Servi
Melissa
M
Marcocci
Claudio
C
Vitti
Paolo
P
Pinchera
Aldo
A
Tonacchera
Massimo
M
D017667:Adipocytes / Q000166:cytology / Q000502:physiology*; D000328:Adult; D000911:Antibodies, Monoclonal; D002454:Cell Differentiation; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D015870:Gene Expression / Q000276:immunology; D006111:Graves Disease / Q000276:immunology / Q000473:pathology / Q000503:physiopathology*; D006801:Humans; D018828:Immunoglobulins, Thyroid-Stimulating / Q000276:immunology; D007150:Immunohistochemistry; D008297:Male; D012333:RNA, Messenger / Q000032:analysis; D011989:Receptors, Thyrotropin / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000911:Antibodies, Monoclonal; D018828:Immunoglobulins, Thyroid-Stimulating; D012333:RNA, Messenger; D011989:Receptors, Thyrotropin; D000242:Cyclic AMP