Sphingosine-1-phosphate mediates fibrosis in orbital fibroblasts in graves’ orbitopathy

Abstract

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 transforming growth factor (TGF)-β and cigarette smoke extract (CSE) on S1P receptor (S1PR) messenger RNAs (mRNA) were evaluated by real-time polymerase chain reaction. 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 h before stimulation with TGF-β, CSE, or interleukin (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 mRNAs 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 pro-fibrotic stimulants upregulated collagen Iα, fibronectin, α-SMA, MMP-1, MMP-2, MMP-9, and TIMP-1 expression in a dose-dependent manner. Conclusion: 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.