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

Sarah E Nicholas,Dimitrios Karamichos,Shrestha Priyadarsini,Tyler G Rowsey,Nawajes A Mandal,Ashley Cowart

doi:10.1371/journal.pone.0182390

Abstract

PurposeTo delineate the role of Sphingolipids (SPLs) in the human cornea and their cross-talks with transforming growth factor beta (TGF-β) in order to develop novel, non-invasive therapies.MethodsHuman 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 I2) and isolated after 4 weeks for further analysis.ResultsData showed that S1P led to a significant decrease in cellular migration where SPHK I2 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 5μM and Sphingosine kinase-2 (SphK2) was also significantly downregulated at concentrations of 0.01μM, 0.1μM, and 5μM; whereas no effects were observed upon stimulation with SPHK I2. S1PR3 was significantly downregulated by 0.1μM and 5μM S1P and upregulated by 5μM and 10μM SPHK I2. Furthermore, both S1P and SPHK I2 regulated corneal fibrosis markers such as alpha-smooth muscle actin, collagen I, III, and V. We also investigated the interplay between two TGF-β isoforms and S1P/SPHK I2 treatments and found that TGF-β1 and TGF-β3 were both significantly upregulated with the 0.1μM S1P but were significantly downregulated with the 5μM S1P concentration. When TGF-β1 was compared directly to TGF-β3 expression, we observed that TGF-β3 was significantly downregulated compared to TGF-β1 in the 5μM concentration of S1P. No changes were observed upon SPHK I2 treatment.ConclusionOur study delineates the role of sphingolipids in the human cornea and highlights their different activities based on the cell/tissue type.

Highlights

Corneal fibrosis, or corneal scarring, is characterized by the emergence of myofibroblasts and excessive deposition of extracellular matrix components (ECM) [1,2,3,4]
Sphingosine kinase-1 (SphK1) was significantly downregulated upon exogenous stimulation with S1P at a concentration of 5μM and Sphingosine kinase-2 (SphK2) was significantly downregulated at concentrations of 0.01μM, 0.1μM, and 5μM; whereas no effects were observed upon stimulation with sphingosine kinase inhibitor 2 (SPHK I2)
We investigated the interplay between two TGF-β isoforms and S1P/SPHK I2 treatments and found that TGF-β1 and TGF-β3 were both significantly upregulated with the 0.1μM S1P but were significantly downregulated with the 5μM S1P concentration

Summary

Introduction

Corneal scarring, is characterized by the emergence of myofibroblasts and excessive deposition of extracellular matrix components (ECM) [1,2,3,4]. This leaves the cornea opaque and can result in partial or complete vision loss [5,6,7,8,9]. S1P has been established as a “growth-like” factor due to its pleiotropic nature and by virtue of their ability to regulate diverse cellular processes, there has been great recent interest in the ability to regulate tissue fibrosis in various organ systems using S1P and/or Cer Studies include numerous organ systems, such as lungs [20], skin [21,22,23,24], liver [25,26,27,28,29], heart [30,31], and eye [11,32,33,34,35]

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