title: Evidence for protein and mRNA TSHr expression in fibroblasts from patients with thyroid-associated ophthalmopathy (TAO) after adipocytic differentiation.
abstract: 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.
authors.affiliation: Dipartimento di Endocrinologia e Metabolismo, Centro Eccellenza AmbiSEN Università di Pisa, Via Paradisa 2, 56124 Pisa, Italy.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Agretti
authors.forename: Giuseppina
authors.initials: G
authors.lastname: De Marco
authors.forename: Melissa
authors.initials: M
authors.lastname: De Servi
authors.forename: Claudio
authors.initials: C
authors.lastname: Marcocci
authors.forename: Paolo
authors.initials: P
authors.lastname: Vitti
authors.forename: Aldo
authors.initials: A
authors.lastname: Pinchera
authors.forename: Massimo
authors.initials: M
authors.lastname: Tonacchera
mesh_terms: 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
chemical_list: D000911:Antibodies, Monoclonal; D018828:Immunoglobulins, Thyroid-Stimulating; D012333:RNA, Messenger; D011989:Receptors, Thyrotropin; D000242:Cyclic AMP