RESUMO
INTRODUCTION: Excessive orbital fibroblast (OF) proliferation and extracellular matrix production, as well as inflammation resulting in the expansion and remodeling of orbital tissue, are characteristic of Graves ophthalmopathy (GO). Our aim was to analyze and inhibit signaling pathways in resident OF that are involved in GO. METHODS/MAIN OUTCOME MEASURES: Primary human OF were obtained from 12 patients with active, severe GO and from 12 healthy control subjects. The cells were characterized by immunofluorescence assay and flow cytometry. Tyrosine phosphorylation of cellular proteins was determined by Western blot techniques, immunoprecipitation, and protein identity with mass spectrometry. Cell proliferation was determined by 5-bromo-2-deoxyuridine incorporation, hyaluronan (HA) production was assessed by a HA-binding protein based assay, and intracellular reactive oxygen species (ROS) were determined by the dichlorofluorescein assay. Clathrin heavy-chain (CHC) expression was inhibited with small interfering RNA technology. RESULTS: Tyrosine phosphorylation of CHC is constitutively increased in vitro in GO-derived OF, independent of serum or other stimulating factors. The proliferative and biosynthetic capabilities (production of HA, ROS) of GO-derived OF are significantly higher than those of OF from healthy control subjects. Down-regulation of CHC expression leads to a normalization of pathologically increased proliferation and production of HA and ROS in GO-derived OFs in vitro. CONCLUSIONS: Our findings strongly suggest that clathrin and clathrin-mediated signaling pathways are involved in the inflammatory signal transduction of OF in GO. With the identification of clathrin, we report a new potential targeting molecule for specific pharmacological inhibition of the local inflammatory response characteristic of GO.