Abstract
Introduction: TSH receptors (TSHR) expressed by differentiated orbital fibroblasts (DOF) may be involved in the pathogenesis of Graves orbitopathy (GO). DOF are able to produce hyaluronan (HA), synthesized by three types of hyaluronan synthases (HAS1; HAS2; HAS3). TSHR stimulation activates the cAMP (adenylyl cyclase) signal transduction pathway. It is unknown whether cAMP activation is involved in HA production.
Aim: The aim of this study was to evaluate the effects of 1) TSH, 2) immunoglobulins from patients with Graves Disease (GD-IgG) or controls (c-IgG) and 3) forskolin (FSK), a direct activator of adenylyl cyclase, on cAMP and HA production and HAS1-3 mRNA expression by DOF.
Methods: Human OF, obtained during decompression surgery from 3 patients with severe GO, were stimulated to differentiate into adipocytes. Differentiation was evaluated by phase-contrast microscopy and TSHR mRNA expression. The differentiated OF were stimulated with rhTSH (10mU/ml), c-IgG (1mg/ml), GD-IgG (1mg/ml) or FSK (50μM). cAMP was measured using a biochemical assay, HAS mRNA expression was measured using qPCR and HA in the supernatant was measured using an ELISA. Results In DOF, expressing higher levels of the TSHR compared with undifferentiated OF, TSH induced a marked cAMP response (2.2-fold compared to control) but no measurable HAS mRNA or HA response. In contrast, FSK induced a 150-fold increase of cAMP, followed by increased HAS1 (150-fold) and HAS2 (2.5-fold) mRNA expression and HA production (4-fold). Stimulation by GD-IgG moderately increased cAMP (1.5-fold) with unaltered HAS mRNA expression and HA levels.
Conclusion: Strong activation of the cAMP pathway by FSK increases the production of HA in differentiated OF. Both TSH and GD-IgG result in a moderately increased cAMP with no measurable changes in HA production. Whether these observations on cAMP-regulated HA synthesis in this in vitro model can be translated to GO awaits further study.