Potentiation of Sphingolipids and TGF-β in the human corneal stroma reveals intricate signaling pathway crosstalks

Corneal haze brought on by fibrosis due to insult can lead to partial or complete vision loss. Currently, corneal transplantation is the gold standard for treating severe corneal fibrosis, which comes with the risk of rejection and the issue of donor tissue shortages. Sphingolipids (SPLs) are known to be associated with fibrosis in various tissues and organs, including the cornea. We previously reported that SPLs are tightly related to Transforming Growth Factor beta (TGF-beta) signaling and corneal fibrogenesis. This study aimed to elucidate the interplay of SPLs, specifically sphingosine-1-phosphate (S1P) signaling, and its' interactions with TGF- beta signaling through detailed analyses of the corresponding downstream signaling targets in the context of corneal fibrosis, in vitro. Healthy human corneal fibroblasts (HCFs) were isolated, plated on polycarbonate membranes, and stimulated with a stable Vitamin C derivative. The 3D constructs were treated with either 5 mu M sphingosine-1-phosphate (S1P), 5 mu M SPHK I-2 (I2; inhibitor of sphingosine kinase 1, one of the two enzymes responsible for generating S1P in mammalian cells), 0.1 ng/mL TGF-beta 1, or 0.1 ng/mL TGF-beta 3. Cultures with control medium-only served as controls. All 3D constructs were examined for protein expression of fibrotic markers, SPLs, TGF-beta s, and relevant downstream signaling pathways. This data revealed no significant changes in any LTBP (latent TGF- beta binding proteins) expression when stimulated with S1P or I2. However, LTBP1 was significantly upregulated via stimulation of TGF-beta 1 and TGF-beta 3, whereas LTBP2 was significantly upregulated only with TGF-beta 3 stimulation. Significant downregulation of TGF- beta receptor II (TGF-beta RII) following S1P stimulation but significant upregulation following I-2 stimulation was observed. Following TGF-beta 1, S1P, and I-2 stimulation, phospho-SMAD2 (pSMAD2) was significantly downregulated. Furthermore, I-2 stimulation led to significant downregulation of SMAD4. Adhesion/proliferation/transcription regulation targets, SRC, FAK, and pERK 1/2 were all significantly downregulated by exogenous S1P, whereas I2 only significantly downregulated FAK. Exogenous TGF-beta 3 caused significant upregulation of AKT. Interestingly, both I-2 and TGF-beta 3 caused significant downregulation of JNK expression. Lastly, TGF-beta 1 led to significant upregulation of sphingosine kinase 1 (SphK1) and sphingosine-1phosphate receptor 3 (S1PR3), whereas TGF-beta 3 caused significant upregulation of only SphK1. Together with previously published work from our group and others, S1P inhibition exhibits great potential as an efficacious anti-fibrotic modality in human corneal stromal ECM. The current findings shed further light on a very complex and rather incompletely investigated mechanism, and cement the intricate crosstalk between SPLs and TGF-beta in corneal fibrogenesis. Future studies will dictate the potential of utilizing SPLs/TGF-beta signaling modulators as novel therapeutics in corneal fibrosis.

Automated summary

Corneal haze caused by fibrosis can lead to vision loss. Corneal transplantation is currently used to treat severe fibrosis, but it has limitations. This study investigates the role of sphingolipids and their interactions with TGF-beta signaling in corneal fibrosis.