Unravelling the interplay of sphingolipids and TGF-β signaling in the human corneal stroma

Purpose To delineate the role of Sphingolipids (SPLs) in the human cornea and their cross-talks with transforming growth factor beta (TGF-beta) in order to develop novel, non-invasive therapies. Methods Human corneal fibroblasts (HCFs) were harvested from healthy donors, stimulated with Vitamin C to promote extracellular matrix assembly, treated with exogenous sphingosine-1-phosphate (S1P) or sphingosine kinase inhibitor 2 (SPHK I-2) and isolated after 4 weeks for further analysis. Results Data showed that S1P led to a significant decrease in cellular migration where SPHK I-2 just delayed it for 24h. Significant modulation of the sphingolipid pathway was also noted. Sphingosine kinase-1 (SphK1) was significantly downregulated upon exogenous stimulation with S1P at a concentration of 5 mu M and Sphingosine kinase-2 (SphK2) was also significantly downregulated at concentrations of 0.01 mu M, 0.1 mu M, and 5 mu M; whereas no effects were observed upon stimulation with SPHK I-2. S1PR3 was significantly downregulated by 0.1 mu M and 5 mu M S1P and upregulated by 5 mu M and 10 mu M SPHK I-2. Furthermore, both S1P and SPHK I-2 regulated corneal fibrosis markers such as alpha-smooth muscle actin, collagen I, III, and V. We also investigated the interplay between two TGF-beta isoforms and S1P/SPHK I-2 treatments and found that TGF-beta 1 and TGF-beta 3 were both significantly upregulated with the 0.1 mu M S1P but were significantly downregulated with the 5 mu M S1P concentration. When TGF-beta 1 was compared directly to TGF-beta 3 expression, we observed that TGF-beta 3 was significantly downregulated compared to TGF-beta 1 in the 5 mu M concentration of S1P. No changes were observed upon SPHK I-2 treatment. Conclusion Our study delineates the role of sphingolipids in the human cornea and highlights their different activities based on the cell/tissue type.