Sphingosine-1-Phosphate Mediates Fibrosis in Orbital Fibroblasts in Graves' Orbitopathy

Purpose: To investigate the effect of sphingosine-1-phosphate (S1P) on fibrosis in orbital fibroblasts in Graves' orbitopathy (GO). Methods: Orbital fibroblasts were cultured from orbital adipose/connective tissues of patients with GO and healthy control subjects. Effects of treatment with TGF-β and cigarette smoke extract (CSE) on S1P receptor (S1PR) messenger RNA (mRNA) and S1P expression were evaluated by real-time polymerase chain reaction and Western blotting. To evaluate the role of S1P in fibrosis, cells were pretreated with W146 (S1PR1 antagonist); JTE013 (S1PR2 antagonist); FTY720 (S1PR1 modulator); or 5C (sphingosine kinase-1 blocker) for 1 hour before stimulation with TGF-β, CSE, or IL-1β. Expression of fibrosis-related proteins (collagen Iα, fibronectin, and α-smooth muscle actin [SMA]) and tissue remodeling-related proteins (matrix metalloproteinases [MMPs] and tissue inhibitor of metalloproteinase [TIMP]-1) was then evaluated by Western blotting. Results: Expression levels of S1PR mRNA and S1P in GO orbital fibroblasts increased upon TGF-β and CSE treatment. Treatment with S1PR blockers and 5C inhibited TGF-β and CSE-induced expression of collagen Iα, fibronectin, and α-SMA, as well as IL-1β-induced expression of MMP-1, MMP-2, MMP-9, and TIMP-1. Exogenous S1P treatment without profibrotic stimulants upregulated collagen Iα, fibronectin, α-SMA, MMP-1, MMP-2, MMP-9, and TIMP-1 expression in a dose-dependent manner. Conclusions: Blocking of S1PR activity and inhibition of S1P synthesis led to decreased expression of fibrosis and tissue remodeling-related proteins in primary cultures of orbital fibroblasts derived from patients with GO. Thus, modulation of S1P activity might have therapeutic potential in the suppression of fibrosis in GO.restrictio