title: A stimulatory thyrotropin receptor antibody enhances hyaluronic acid synthesis in graves' orbital fibroblasts: inhibition by an IGF-I receptor blocking antibody.
abstract: BACKGROUND
  Graves' ophthalmopathy (GO) is characterized by expanded volume of the orbital fat and extraocular muscle tissues and elevated levels of TSH receptor autoantibodies (TRAb). The expansion of orbital tissues involves accumulation of hyaluronic acid (HA) within the orbit.
  
  
  OBJECTIVE
  The objective of the study was to determine whether a monoclonal stimulatory TRAb (M22) impacts HA synthesis in GO orbital cells and, if so, whether this might be blocked by an IGF-I receptor (IGF-IR)-blocking antibody (1H7) or inhibitors of various downstream signaling cascades.
  
  
  METHODS
  GO orbital fibroblast cultures (n = 6) were treated with M22, bovine TSH (bTSH), or IGF-I in serum-free medium. Some cultures also received 1H7, LY294002, rapamycin, or protein kinase A inhibitor.
  
  
  METHODS
  HA production and phosphorylated Akt levels in media or immunoblotting for phosphorylated Akt were measured.
  
  
  RESULTS
  M22 or bTSH stimulated HA synthesis (2.1-fold with 100 ng/ml M22 and 1.9-fold with 10 U/liter bTSH; P < 0.05 each). M22-induced HA synthesis was inhibited by LY294002 or rapamycin but not by protein kinase inhibitor. HA synthesis stimulated by M22 or IGF-I was inhibited by 1H7 (mean 36.6 ± 5.6% and mean 45.8 ± 7.6%, respectively; P < 0.05 each). Similarly, M22- or IGF-I-stimulated Akt phosphorylation was inhibited by 1H7 (mean 54 ± 9.6 and 36.1 ± 8.8%, respectively; P = 0.01 each).
  
  
  CONCLUSIONS
  The stimulatory TRAb M22 increases HA production in undifferentiated GO orbital fibroblasts via phosphoinositide 3-kinase/phosphorylated AKT/mammalian target of rapamycin activation. Blockade of IGF-IR inhibits both HA synthesis and Akt phosphorylation induced by M22 or IGF-I in these cells, suggesting that TSH receptor and IGF-IR signaling may be closely linked in the GO orbit.
authors.affiliation: Division of Pediatric Endocrinology and Metabolism, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55902, USA.
authors.forename: Seema
authors.initials: S
authors.lastname: Kumar
authors.forename: Seethalakshmi
authors.initials: S
authors.lastname: Iyer
authors.forename: Hilary
authors.initials: H
authors.lastname: Bauer
authors.forename: Michael
authors.initials: M
authors.lastname: Coenen
authors.forename: Rebecca S
authors.initials: RS
authors.lastname: Bahn
mesh_terms: D019138:Antibodies, Blocking / Q000378:metabolism / Q000494:pharmacology*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D049970:Graves Ophthalmopathy / Q000276:immunology*; D006801:Humans; D006820:Hyaluronic Acid / Q000096:biosynthesis*; D017526:Receptor, IGF Type 1 / Q000037:antagonists & inhibitors / Q000276:immunology*; D011989:Receptors, Thyrotropin / Q000276:immunology*; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology
chemical_list: D019138:Antibodies, Blocking; D011989:Receptors, Thyrotropin; D006820:Hyaluronic Acid; D017526:Receptor, IGF Type 1