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Unravelling the Interplay of Sphingolipids and Tgf-β Signaling in the Human Corneal Stroma

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Unravelling the Interplay of Sphingolipids and TGF-β signaling in the human corneal stroma
Corneal fibrosis occurs after an incidence of injury to the corneal surface. This is characterized by emergence of myofibroblasts and an irregular distribution of extracellular matrix components. Sphingolipids have been linked to fibrosis in various tissues and organs in the past but little is known about their roles in the human cornea and corneal fibrosis. In our study, we determined the effects of sphingosine-1-phosphate (S1P) on human corneal fibroblasts (HCF) using an established 3D self-assembled in vitro model. Data showed that S1P led to a significant decrease in cellular migration where Sphingosine kinase inhibitor 2 (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 Iregulated 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 Itreatment. Our study delineates the role of sphingolipids in the human cornea and highlights their different activities based on the cell/tissue type.
Sphingolipids; Corneal fibrosis; TGF-β; S1P; SPHK I2; Cellular Migration; S1PR3; Eye.

  1. Introduction

Corneal fibrosis, or corneal scarring, is characterized by the presence of myofibroblasts and excessive deposition of extracellular matrix (ECM) components (Beales, Funderburgh et al. 1999Fini 1999Zieske 2001Funderburgh, Mann et al. 2003). This leaves the cornea opaque and can result in partial or complete vision loss (Chirila 2001Niederkorn 2003Baradaran-Rafii A 2007Guo, Hutcheon et al. 2007Coster, Jessup et al. 2009). Currently, more than 10 million people worldwide are blind because of corneal scarring and approximately 100 million suffer from impaired vision. The mechanics of fibrosis have been studied for years but there are currently no available drugs for scarring treatment. Recently the role of sphingolipids (SPLs) has been linked to fibrosis in a variety of tissues and organs (Watterson, Lanning et al. 2007Swaney, Moreno et al. 2008Shea and Tager 2012Takuwa, Okamoto et al. 2012).
Bioactive SPLs most notably Sphingosine-1-phosphate (S1P) and ceramide (Cer), are now recognized to be important mediators of many basic cellular processes such as cell migration, survival, contraction, proliferation, gene expression, and cell-cell interactions(Shea and Tager 2012). The impact of SPLs in human diseases associated with inflammation, neovascularization, tumorigenesis, and diabetes have been recognized but are still understudied(Levade, Malagarie-Cazenave et al. 2002Riboni, Bassi et al. 2002Riboni, Campanella et al. 2002Buccoliero and Futerman 2003Litvak, Bilchik et al. 2003Brush, Tran et al. 2010). S1P has been established as a growth-like factor due to its pleiotropic nature and therefore, by virtue of their ability to regulate diverse cellular processes, there has been substantial recent interest in the ability to regulate tissue fibrosis in various organ systems using S1P and/or Cer (Roger A. Sabbadini, 2010). S1P has been studied more extensively than Cer in regards to tissue fibrosis. Studies include numerous organ systems, such as lungs (Shea, Brooks et al. 2010), skin (Sauer, Vogler et al. 2004Xin, Ren et al. 2004Radeke, von Wenckstern et al. 2005Bu, Kapanadze et al. 2008), liver (Davaille, Li et al. 2002Ikeda, Watanabe et al. 2009Li, Jiang et al. 2009Li, Zheng et al. 2011Liu, Yue et al. 2011), heart (Gellings Lowe, Swaney et al. 2009Takuwa, Ohkura et al. 2010), and eye (Swaney, Moreno et al. 2008Caballero, Swaney et al. 2009Rao 2014Simon, Prado Spalm et al. 2015Priyadarsini, McKay et al. 2016).
Interestingly, the role of S1P in fibrosis is somewhat controversial. It was originally characterized as a potent stimulator of fibroblast proliferation in Swiss 3T3 cells (Zhang, Desai et al. 1991). S1P has also been shown to inhibit the proliferation of hepatic myofibroblasts (Davaille, Gallois et al. 2000) and in human epidermal keratinocytes (Kim, Kim et al. 2004). In the lungs, S1P signaling through sphingosine-1-phosphate receptor 1 S1P1 appears to protect against the development of fibrosis. Conversely, S1P appears to promote fibrosis in other organ systems  (Shea and Tager 2012) (skin, liver, heart, retina) likely through activation of transforming growth factor-β (TGF-β) signaling pathways and/or by promoting fibroblast migration.
Surprisingly, very little is known about the role of SPLs in the human cornea and the mechanisms of corneal fibrosis. In fact, there are only two reports that showed the presence of Sphingosine kinase-1 (SphK1), Sphingosine kinase-2 (SphK2), S1P1-3,5 receptor proteins (Swaney, Moreno et al. 2008) and mRNA (Qi, Priyadarsini et al. 2017) in cultured human primary corneal fibroblasts. Expression of S1P receptor’s mRNA have also been noted by (Watsky, Weber et al. 2010), in cultured corneal epithelial cells mimicking wound healing responses in vivo.
In this study, using our established 3D in vitro model, for the first time we investigated the molecular involvement of S1P in human corneal fibroblasts (HCFs) and the interplay between S1P and TGF-β isoforms. Delineating the role of SPLs in the human cornea, might pave the way for novel therapeutic agents.

  1. Materials and Methods
    1. . Human Corneal Fibroblast cultures

Primary HCFs were isolated and cultured from healthy human corneas obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA). All research adhered to the tenets of the Declaration of Helsinki. Briefly, corneal epithelium and endothelium were scrapped and removed from donor corneas. Stromal tissue cut into 2x2mm pieces was placed into T25 flasks and allowed to adhere. Explants were cultured with Eagle’s Minimum Essential Medium (EMEM: ATCC; Manassas, VA) containing 10% fetal bovine serum (FBS: Atlantic Biological’s; Lawrenceville, CA) and 1% antibiotic (Gibco® Antibiotic-Antimycotic: Life Technologies; Grand Island, NY). The cells were allowed to grow to 100% confluence at 37°C, 5% CO2. Passages 5 – 7 were used throughout the experiments.
2.2. Construct Assembly
HCFs were plated on six-well size polycarbonate membrane inserts with 0.4-μm pores (Transwell; Corning Costar; Charlotte, NC) at a density of 1×106 cells/ml (Karamichos, Lakshman et al. 2009Karamichos, Guo et al. 2010Karamichos, Hutcheon et al. 2011Karamichos, Zareian et al. 2012).The cells were cultured in EMEM with 10% FBS and stimulated with a stable Vitamin C derivative (0.5mM 2-O-α-d-glucopyranosyl-l-ascorbic acid: American Custom Chemicals Corporation; San Diego, CA). Cultures were grown for 4 weeks before further processing. Cultures without any treatment served as the controls (C), and fresh media were supplied every other day for the duration of the experiment. Three different groups were tested: (1) Control (C): EMEM+FBS+VitC (construct medium); (2) S1P: construct medium+S1P at 0.01μM, 0.1μM, or 5μM (Wendler and Rivkees 2006Watterson, Lanning et al. 2007); and (3) SPHK I2: construct medium+SPHK I2 at 2μM, 5μM, and 10μM. A stock solution of S1P (Avanti Polar Lipids; Alabaster, AL) was made at a concentration of 125µM for all S1P treatments by dissolving S1P powder in 4mg/ml of BSA in water at 37˚C in a glass vessel. SPHK I2 (Cayman Chemicals, USA) is a selective inhibitor of SphK1 (French, Schrecengost et al. 2003) and a stock solution was made at a concentration of 5mM by dissolving the powder in DMSO. All experimental groups were repeated at least three times before processed for protein expression using Western Blot analysis.
2.3. Western blot
Western blot analyses were performed on all constructs and their protein concentrations and purities were determined via Pierce™ BCA Protein Assay (ThermoFisher Scientific; Rockford,IL). Precast Novex 4-20% Tris-Glycine Mini Gels (Life Technologies; Carlsbad, CA) were used for gel electrophoresis and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc; Hercules, CA). After a one hour incubation in a 5% BSA blocking solution (ThermoScientific; Rockford, IL), the membranes were incubated with primary rabbit polyclonal antibodies: anti-TGF-β1 (Abcam; Cambridge, MA), anti-TGF β3 (Abcam; Cambridge, MA), anti-SphK1 (Abcam; Cambridge, MA), anti-SphK2 (Abcam; Cambridge, MA), anti-EDG3 (S1PR3; Abcam; Cambridge, MA), anti-Collagen I (Abcam; Cambridge, MA), anti-Collagen III (Abcam; Cambridge, MA), anti-Collagen V (Abcam; Cambridge, MA), and anti-alpha smooth muscle Actin (Abcam; Cambridge, MA) at 1:1000 dilutions overnight at 4°C. Subsequently, the membranes were washed and incubated with a secondary antibody (Alexa Flour® 568 Donkey anti-Rabbit, IgG (H+L); Life Technologies; Carlsbad, CA) at 1:2000 dilutions for one hour. The UVP imaging system (VisionWorks™LS Image Acquisition & Analysis Software; Cambridge, UK) was used for signal detection. The results were analyzed by normalizing the values to that of the house keeping antibody, anti-beta Actin (Abcam; Cambridge, MA) expression, and the fold expression was plotted.
2.4. Cellular migration
Cell migration was measured using an in vitro 2D scratch assay method (Liang, Park et al. 2007) where HCFs were seeded into 6-well plates at a density of 1×106 cells/ml and cultured in the following: (1) Control (C): construct medium; (2) S1P : construct medium+S1P at 0.01μM, 0.1μM, or 5μM; and (3) SPHK I2: construct medium+ SPHK I2 at 2μM, 5μM, and 10μM. After a 24-hour incubation period, allowing the cells to adhere, a scratch was administered through the confluent cell layer using a pipette tip. An AmScope IN200TA-M Digital Long Working Distance Inverted Trinocular Microscope (Irvine, CA) was used to capture images of each scratch wound which was repeated at intervals of 0, 4, 24, and 48 hours to monitor wound closure. Cell migration was measured and quantified using ImageJ software.
2.5. Statistical Analysis
All experiments were repeated at least 3 times and the statistical significance was performed by ANOVA and/or non-parametric t test analysis, where p<0.05 was considered to be statistically significant. Graph Pad Prism 6 software was used for all statistical analysis.

  1. Results

3.1. Effects of S1P and SPHK I2 on cellular migration
Studies have shown that corneal fibroblasts and myofibroblasts modulate important aspects of cell behavior such as adhesion, migration, and differentiation (Wilson 2012Gallego-Munoz, Ibares-Frias et al. 2016) . We investigated and quantified cell migratory pattern for HCFs when stimulated with both S1P (Fig. 1B) and SPHK I2 (Fig. 1C), at various concentrations compared to controls (Fig. 1A). Using an in vitro scratch assay model we observed decreased cellular migration with all S1P concentrations (Fig. 1D). The highest concentration tested here, 5µM S1P, showed the most severe and significant decrease at both 24 and 48h time points (p≤0.0001). At 24h and 48h, cellular migration with 5µM S1P stimulated cells only closed 14% of the wound when compared to 100% with Controls.
Similar pattern was observed with SPHK I2 stimulated cells (Fig. 1E). At 24h, controls had  closed the wound (100%) where cells stimulated with 10uM SPHK Ionly managed to close 44% (p≤0.01) of the wound indicating severe interruption of cellular migration patterns by SPHK I(Fig. 1C), similar to S1P (Fig. 1B), however, at the 48h time point the wound had fully closed.
3.2. Effects of S1P and SphKI2 on sphingolipid-related pathways
S1P is formed by active SphK (Hait, Sarkar et al. 2005Maceyka, Alvarez et al. 2008) and signals through S1P receptors 1-5 which preferentially couple to different downstream pathways. The downstream pathways of S1PR1-S1PR5 are reportedly overlapping yet functionally oppositional; with various intracellular targets that are known to engage in signal processing (Hyder, Kemppainen et al. 2015). S1P  is known to stimulate cell proliferation and migration in human aortic endothelial cells (Kimura, Watanabe et al. 2000), murine satellite cells (Calise, Blescia et al. 2012), WiT49 cells (Li, Sanchez et al. 2008), human thyroid cancer cells (Balthasar, Samulin et al. 2006), and many other cell types and can be inhibited by SPHK Iwhich is a selective inhibitor of SphK1 with anti-proliferative capacity (French, Schrecengost et al. 2003). We therefore investigated the role of S1P and SPHK Iin HCFs, using our in vitro model.
Exogenous S1P stimulation led to significant downregulation of SphK1 protein expression (Fig. 2A; p≤ 0.0001) at 5μM concentration whereas of SphK2 was significantly downregulated at 0.01µM (Fig. 3; p≤0.01), 0.1µM (Fig. 3; p≤0.0001), and 5µM (Fig. 3; p≤0.0001) concentrations (Fig. 2B). Interestingly, exogenous SPHK Ihad no significant effects on SphK1 or SphK2 across all the concentrations tested here (2µM, 5µM, and 10µM).
In addition, Figure 2C shows significant downregulation of sphingosine-1-phosphate receptor 3 (S1PR3) protein expression. S1PR3 is one of five G-protein-coupled receptors that bind S1P and transactivates downstream signaling pathways. S1PR3 downregulation was observed following stimulation with S1P at concentrations of 0.1µM (p≤0.0001) and 5µM (p≤0.0001), whereas in Figure 2D we detected significant upregulation of S1PR3 upon stimulation with 5µM (p≤0.01) and 10µM (p≤0.01) concentrations of SPHK I2.
3.3. Effects of S1P and SPHK Ion fibrosis (assembly)
We determined the effects of both S1P and SPHK Ias they relate to corneal fibrosis and fibrotic markers in HCFs. In Figure 3A shows significant downregulation was observed (p≤0.0001) in α-SMA protein expression upon stimulation with only 5µM S1P with no changes observed when constructs were stimulated with SPHK I(Figure 3B).
Collagen I was significantly downregulated (Figure 4A; p≤0.0001) when constructs were stimulated with 5µM S1P but no change when constructs were stimulated with SPHK I(Figure 4B). Collagen V was significantly downregulated (Figure 4C; p≤0.0001) upon stimulation with 5µM S1P and significantly upregulated (Figure 4D; p≤0.01) with 10µM SPHK Istimulation.
We observed no significant changes of collagen III upon S1P stimulation (Figure 4E), while SPHK Iexogenous stimulation led to significant downregulation (2µM; p≤0.001, 5µM; p≤0.01, and 10µM; p<0.01) across all the concentrations tested here (Figure 4F).
When Collagen I/III ratio was analyzed, stimulation with 5µM S1P led to significant downregulation (Figure 5A; p≤0.0001) where significant upregulation (Figure 5B; p<0.01) was observed upon stimulation with only 2µM SPHK Iand an upward trend in the higher concentrations. Collagen I/V ratio was unaffected following S1P stimulation (Figure 5C) where a downward trend was observed through the SPHK Itreatment concentration series with significance (Figure 5D; p<0.001) observed in the 10µM concentration only.
3.4. S1P and SPHK Isignaling interactions with TGF-β isoforms
A number of studies have linked S1Ps fibrotic activity with TGF-β1. In this study, we investigated the interplay between S1P/ SPHK I2 and the two TGF-β isoforms TGF-β1 and TGF–β3. In our previous studies, as well as others, have shown that TGF-β3 has antifibrotic properties (Karamichos, Hutcheon et al. 2011).
Figure 6 shows similar results when comparing TGF-β1 and TGF-β3 expression, following exogenous S1P stimulation. TGF-β1 showed significant (p≤0.01) upregulation following S1P stimulation at 0.1µM concentration, where significant (p≤0.0001) downregulation was observed at 5µM concentration (Fig. 6A). Similarly, TGF-β3 expression was significantly (p≤0.01) upregulated at 0.1µM S1P and significantly (p≤0.0001) downregulated at 5µM S1P concentration (Fig. 6B). TGF-β1 and TGF-β3 expression did not change following exogenous stimulation with SPHK I2.
When TGF-β1 was compared directly to TGF-β3 expression, following S1P stimulation, we found that only at 5µM S1P there was a significant (p≤0.0001) downregulation in TGF-β3 when compared to TGF-β1. In a similar comparison, for the SPHK Istimulated constructs, no differences were observed between TGF-β1 and TGF-β3 at any of the concentrations tested here.

  1. Discussion

Human corneal fibrosis is a usual outcome following corneal injury. Despite significant advancements in the field, current treatments are limited. One of the problems is that corneal wound healing is a complex process, involving corneal cells, ECM components, and growth factors. The mechanisms by which corneal fibrosis can be prevented, or even reversed, remains elusive. The depth of the problem is clear when we consider that a total of 250 million people worldwide have compromised vision and around 6 million have been blinded, majorly due to corneal fibrosis and scarring.
The main characteristic of a corneal scar is the presence of myofibroblasts, often indicated by the expression of α-SMA, and the excessive and improper deposition of ECM components such as collagen III. Preventing scar formation would be ideal; however, studies investigating the development of non-fibrotic healing in human corneas are extremely limited. There are two main reasons for this: 1) Unavailability of human tissue makes investigations difficult, and 2) Mechanistic pathways of corneal scarring are not fully understood.
In this study, we investigated the role of SPLs in corneal fibrosis, using an in vitro model, and how these are interacting with a major player in the human cornea; TGF- β. SPLs have been recently linked to fibrosis in a variety of tissues and they are found to be in close connection to the TGF-β pathway. TGF-β is a superfamily of pleiotropic cytokines that exist as five isoforms, three of which have been identified in humans: TGF-β1, TGF-β2, and TGF-β3. The three isoforms have shown to affect a variety of biological processes which include cellular proliferation, differentiation, and migration in most cells. In our previous studies, we found that while TGF-β1 exhibited profibrotic effects in HCFs, TGF-β3 displayed anti-fibrotic capabilities. Most notably, TGF-β3 was able to stimulate HCFs to secrete larger amounts of ECM, while maintaining anti-fibrotic characteristics and exhibited a “rescuing” effect in vitro (Karamichos, Hutcheon et al. 2014).
The cross-talk between S1P and TGFβ has been appreciated through several recent studies (Watterson, Lanning et al. 2007Shea and Tager 2012Takuwa, Ikeda et al. 2013). Briefly, it has been shown that TGFβ up-regulates Sphk1 mRNA and protein levels and increases SPHK1 activity in dermal fibroblasts (Yamanaka, Shegogue et al. 2004). In contrast, TGFβ reduced S1P phosphatase activity. Interestingly, down-regulating Sphk1 expression blocked TGFβ-mediated increases in TIMP-1 protein. It was therefore proposed that SPHK1 is a downstream mediator for the induction of TIMP1 by TGFβ (Yamanaka, Shegogue et al. 2004). It has also been demonstrated that S1P utilizes its receptor signaling to stimulate phosphorylation and activate TGFβRI kinase, resulting in phosphorylation of Smad2 and Smad3, independently of the TGFβ ligand, leading to keratinocyte proliferation and migration (Sauer, Vogler et al. 2004). S1P-stimulated Smad phosphorylation was inhibited by down-regulation of TGFβ receptor type II and also by the S1P3 antagonist, suramin, suggesting that S1P transactivates TGFβRII in an S1P3-dependent process (Xin, Ren et al. 2004). Consistent with these observations, another study showed that matrix remodeling in response to acute liver injury was abrogated in S1P2-/- mice, which was attributed to reduced accumulation of myofibroblasts, as shown by a lower induction of a-SMA, TIMP1, TGFβ, and PDGF, suggesting that a-SMA induction by S1P may be S1P2– and Rho-dependent (Serriere-Lanneau, Teixeira-Clerc et al. 2007).
In the cornea, TGF-β is well studied whereas very little is currently known about the effects of S1P and its interrelations with the TGF-β pathway. Our study suggests that exogenous supply of S1P to HCFs downregulate SphK1, SphK2, and S1PR3 and had an effect on assembly by downregulating collagens I, V, and I/V ratio. We also observed downregulation of α-SMA and decreased cellular migration, which could indicate that S1P may have anti-fibrotic capabilities in the human cornea. S1P had the same effect on TGF-β1 and TGF-β3, where we observed upregulation with the 0.1µM concentration but downregulation with the 5µM concentration. Upon observation of SPHK Itreatment, S1PR3 and collagens V and I/III ratio were upregulated where collagens III and I/V ratio were downregulated. This could indicate that SPHK Imay aid in ECM remodeling. Additionally, SPHK Iseems to inhibit endogenous sources of S1P within the cell which signal for cellular actin mobilization thus causing decreased cellular migration. Our current findings suggest that S1P likely play important roles as both primary and secondary signaling messengers in the human cornea. Exogenous S1P is known to act as an important second messenger inside the cell (Spiegel and Milstien 2000) but most of its influences on cell signaling are reportedly due to cell surface receptor binding (Payne, Milstien et al. 2002Watterson, Sankala et al. 2003Anliker and Chun 2004Wendler and Rivkees 2006).
Stimulation with S1P appears to induce or inhibit cell migration based on the tissue and cell type (cell-type specific). In our study, we observed that exogenous stimulation with S1P inhibits corneal cell migration and we suggest that S1P is acting through S1PR3. It is reported in the following studies that S1P induced cell migration is mediated through S1PR1 and S1PR3. Our data revealed that S1P significantly downregulated S1PR1 and S1PR3 mRNA expression (p ≤ 0.05), further indicating that S1P inhibits cell migration. Similarly, Wendler and co-authors observed reduced cell migration and inhibited mesenchymal cell formation in AV canal cushion tissue with increasing concentrations of S1P treatment. They suggested S1P is necessary for early heart development (Wendler and Rivkees 2006). Becciolini et al. 2006 reported the anti-migratory actions of S1P in C2C12 myoblasts; stating that S1P inhibited cell migration and abolished chemotactic responses to IGF-1(Becciolini, Meacci et al. 2006). Arikawa et al. 2003 investigated the effects of S1P on migration of B16 melanoma cells. They found that S1P treatment led to inhibited migration of S1PR1 and S1PR3-overexpressing cells. They believe that their data indicates that S1PR2 mediates S1P inhibition of migration, whereas S1PR1 and S1PR3 mediate S1P stimulation of cell migration (Arikawa, Takuwa et al. 2003). Kawa et al. 1997 found that S1P decreased trans-endothelial migration and invasiveness of neutrophils into human umbilical vein endothelial cells (HUVEC)-covered collagen layers. They suggest that S1P acts as a specific and effective motility regulator that could be used to mediate invasive migration of neutrophils (Kawa, Kimura et al. 1997).
Contrastingly, Simón MV et al. 2015 (Simon, Prado Spalm et al. 2015) observed that treatment with S1P enhanced Müller glial cell migration where SPHK I2 nearly prevented glial migration. They suggest that Müller glial cells synthesize S1P, which signals through S1PR3 to induce glial migration. Esche et al. 2010 also investigated the effects of S1P on Müller glial cell migration and observed slightly induced migration (Esche, Hirrlinger et al. 2010). Li et al. 2011 examined the effects of S1P in the human hepatic myofibroblasts (hMFs) and determined that S1P exerted powerful migratory action on hMFs where S1PR1 and S1PR3 were strongly induced and S1PR2 was strongly inhibited (Li, Zheng et al. 2011). Liu et al. 2011 investigated the functions of S1P in human hepatic stellate cells (HSC) line, LX-2 cells and found that S1P exerted powerful migratory action on LX-2 cells; S1PR1 and S1PR3 inducing migration and S1PR3 inhibiting migration (Liu, Yue et al. 2011). Maceyka et al. 2008 determined that S1P promoted cell migration through S1PR1 in human embryonic kidney cells (HEK 293), transfected with siRNA targeted to SphK (Maceyka, Alvarez et al. 2008). Kimura et al. 2000 studied the effects of S1P on human aortic endothelial cell proliferation and migration and showed that S1P induced both proliferation and migration (Kimura, Watanabe et al. 2000). Previous independent studies also report the stimulation of endothelial cell migration by S1P (Hisano, Yatomi et al. 1999Lee, Kim et al. 1999Wang, Van Brocklyn et al. 1999Osada, Yatomi et al. 2002Osborne and Stainier 2003McVerry and Garcia 2004).
Van Brocklyn, JR, 2010 published a review on the effects of S1P on cancer cell migration. He states that the effects of S1P on cell migration vary between different cancers and even among different models of the same cancer type, in some cases inducing migration and inhibiting in others (Brocklyn 2010). The literature supports that some S1P receptors have opposite effects on cells/tissues (Sabbadini 2011). S1PR2 has been reported to have anti-migratory effects whereas; S1PR1 and S1PR3 are pro-migratory in B16 melanoma cells (Arikawa, Takuwa et al. 2003), Chinese hamster ovary cells (Kon, Sato et al. 1999), human umbilical vein endothelial cells(Paik, Chae et al. 2001), mouse embryonic endothelial cells (MEECs)(Chae, Paik et al. 2004), bovine aortic endothelial cells (BAECs) (Langlois, Gingras et al. 2004), human gastric cancer cell lines (Yamashita, Kitayama et al. 2006), and in human hepatic myofibroblasts(Li, Zheng et al. 2011) (hMFs).
Kimura et al. 2000 and Wendler C, Rivkees SA, 2006 report oppositional effects of S1P in the heart as we’ve observed in the eye; oppositional effects in the human cornea and in the retina. Means CK, Brown JH, 2009 published a review on S1P receptor signaling in the heart where they mention, based on the literature, it is known that S1P inhibits migration of smooth muscle cells whereas it promotes migration of endothelial cells (Alewijnse, Peters et al. 2004Means and Brown 2009). These findings are very interesting as our study suggests that S1P inhibits cell migration in HCFs whereas it promotes migration in the retina (Simon, Prado Spalm et al. 2015). The eye, like the heart, is a complex organ with various cell types that appear to respond differently to S1P. As we have just scratched the surface of S1P signaling in the cornea, further investigations into the downstream signaling effects and interplay with TGF-β are necessary for the future potential design of novel therapeutics targeting S1P signaling in the opportune timeframe post incidence of corneal fibrosis to successfully manage ECM remodeling.

  1. Conclusions

The role of S1P in corneal biology is still unclear. Further elucidation of how S1P modulates TGF-β signaling may lead to the discovery of pro-fibrotic pathways that may be altered to reduce corneal fibrosis.

Authors’ contributions

S.E.N. conducted experiments, analyzed data, and wrote the manuscript. T.G.R. conducted experiments and analyzed data. S.P. conducted experiments. N.A.M. reviewed/edited the manuscript. D.K. conceived the project, provided reagents and supplies, and reviewed/edited the manuscript.

Conflict of interest

The authors declare that no conflict of interest exists.
This work was supported by the National Institutes of Health Grants 5R01EY023568 (DK), 5R01EY020886 (DK) and R21-EY025256-01.
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