Thyrotropin Receptor-Stimulating Graves' Disease Immunoglobulins Induce Hyaluronan Synthesis by Differentiated Orbital Fibroblasts from Patients with Graves' Ophthalmopathy Not Only Via Cyclic Adenosine Monophosphate Signaling Pathways

Abstract

Both expression of the thyrotropin receptor (TSHR) and the production of hyaluronan (HA) by orbital fibroblasts (OF) have been proposed to be implicated in the pathogenesis of Graves' ophthalmopathy (GO). HA is synthesized by three types of HA synthase. We hypothesized that TSHR activation by recombinant human TSH (rhTSH) and TSHR-stimulating Graves' disease immunoglobulins (GD-IgGs) via induced cyclic adenosine monophosphate (cAMP) signaling increases HA synthesis in differentiated OF from GO patients. Cultured human OF, obtained during decompression surgery from 17 patients with severe GO, were stimulated in vitro to differentiate into adipocytes. Differentiation was evaluated by phase-contrast microscopy. The differentiated OF were stimulated by rhTSH or by TSHR-stimulating GD-IgG. We measured cAMP using a biochemical assay, HA synthase mRNA expression by quantitative polymerase chain reaction, and HA in the supernatant by enzyme-linked immunosorbent assay. All differentiated OF cultures expressed higher levels of TSHR mRNA than nondifferentiated OF cultures. Stimulation by rhTSH induced a marked cAMP response in 11 of 12 differentiated OF cultures, but no measurable HA response in all but one differentiated OF cultures. By contrast, stimulation by GD-IgG induced a moderate cAMP response in a number of differentiated OF cultures, but a marked HA response in the majority of differentiated OF cultures. Stimulation of differentiated OF by GD-IgG, but not by rhTSH, induces HA synthesis in the majority of patients, suggesting that in most patients TSHR-mediated cAMP signaling does not play a pivotal role in GD-IgG-induced HA synthesis in differentiated OF cultures.