TY - JOUR
T1 - Analysis of sphingolipids in human corneal fibroblasts from normal and keratoconus patients
AU - Qi, Hui
AU - Priyadarsini, Shrestha
AU - Nicholas, Sarah E.
AU - Sarker-Nag, Akhee
AU - Allegood, Jeremy
AU - Chalfant, Charles E.
AU - Mandal, Nawajes A.
AU - Karamichos, Dimitrios
PY - 2017
Y1 - 2017
N2 - The pathophysiology of human keratoconus (KC), a bilateral progressive corneal disease leading to protrusion of the cornea, stromal thinning, and scarring, is not wellunderstood. In this study, we investigated a novel sphingolipid (SPL) signaling pathway through which KC may be regulated. Using human corneal fibroblasts (HCFs) and human KC cells (HKCs), we examined the SPL pathway modulation. Both cell types were stimulated by the three transforming growth factor (TGF)-β isoforms: TGF-β1 (T1), TGF-β2 (T2), and TGF-β3 (T3). All samples were analyzed using lipidomics and real-time PCR. Our data showed that HKCs have increased levels of signaling SPLs, ceramide (Cer), and sphingosine 1-phosphate (S1P). Treatment with T1 reversed the increase in Cer in HKCs and treatment with T3 reversed the increase in S1P. S1P3 receptor mRNA levels were also significantly upregulated in HKCs, but were reduced to normal levels following T3 treatment. Furthermore, stimulation with Cer and S1P led to significant upregulation of fibrotic markers in HCFs, but not in HKCs. Additionally, stimulation with a Cer synthesis inhibitor (FTY720) led to significant downregulation of specific fibrotic markers in HKCs (TGF-β1, collagen type III, and α smooth muscle actin) without an effect on healthy HCFs, suggesting a causative role of Cer and S1P in fibrogenesis. Overall, this study suggests an association of the SPL signaling pathway in KC disease and its relation with the TGF-β pathway.
AB - The pathophysiology of human keratoconus (KC), a bilateral progressive corneal disease leading to protrusion of the cornea, stromal thinning, and scarring, is not wellunderstood. In this study, we investigated a novel sphingolipid (SPL) signaling pathway through which KC may be regulated. Using human corneal fibroblasts (HCFs) and human KC cells (HKCs), we examined the SPL pathway modulation. Both cell types were stimulated by the three transforming growth factor (TGF)-β isoforms: TGF-β1 (T1), TGF-β2 (T2), and TGF-β3 (T3). All samples were analyzed using lipidomics and real-time PCR. Our data showed that HKCs have increased levels of signaling SPLs, ceramide (Cer), and sphingosine 1-phosphate (S1P). Treatment with T1 reversed the increase in Cer in HKCs and treatment with T3 reversed the increase in S1P. S1P3 receptor mRNA levels were also significantly upregulated in HKCs, but were reduced to normal levels following T3 treatment. Furthermore, stimulation with Cer and S1P led to significant upregulation of fibrotic markers in HCFs, but not in HKCs. Additionally, stimulation with a Cer synthesis inhibitor (FTY720) led to significant downregulation of specific fibrotic markers in HKCs (TGF-β1, collagen type III, and α smooth muscle actin) without an effect on healthy HCFs, suggesting a causative role of Cer and S1P in fibrogenesis. Overall, this study suggests an association of the SPL signaling pathway in KC disease and its relation with the TGF-β pathway.
KW - Cornea
KW - Lipidomics
KW - Transforming growth factor-β
UR - http://www.scopus.com/inward/record.url?scp=85016595407&partnerID=8YFLogxK
U2 - 10.1194/jlr.M067264
DO - 10.1194/jlr.M067264
M3 - Article
C2 - 28188148
AN - SCOPUS:85016595407
SN - 0022-2275
VL - 58
SP - 636
EP - 648
JO - Journal of Lipid Research
JF - Journal of Lipid Research
IS - 4
ER -