Abstract
Purpose :
Secreted Ly-6/uPAR Related Protein-1 (SLURP1), abundantly expressed by the corneal epithelium (CE) and secreted to the tear film, is an anti-inflammatory protein with anti-tumor properties. TGF-b pathway has diverse roles in tissue homeostasis as well as pathological conditions including epithelial-mesenchymal transition (EMT) during tumor metastasis. Here, we studied the influence of SLURP1 on canonical TGF-b signaling using Slurp1X-/- mouse cornea and human corneal limbal epithelial (HCLE) cells.
Methods :
The effect of Slurp1 ablation on TGF-b signaling was studied by comparing TGF-b Receptor 2 (TGFBR2), Smad4, and phospho-Smad2/3 expression in wild type (WT) and Slurp1X-/- mouse corneas by immunoblots and immunofluorescent staining. The effect SLURP1 on TGF-b signaling was analyzed by comparing the TGF-b-induced Smad2/3 phosphorylation and E-cadherin expression in HCLE and HCLE-SLURP1 cells engineered to over-express SLURP1.
Results :
Slurp1X-/- mouse corneas displayed elevated expression (127% of WT) and prominent nuclear localization of TGFBR2, coupled with elevated expression of Smad4, and phospho-Smad2/3. Upon stimulation with 5ng/ml TGF-b, HCLE-SLURP1 displayed decreased SMAD2/3 phosphorylation compared with that in HCLE cells. After 1h of TGF-b addition, the phospho-SMAD2/Total SMAD2 ratio was 20% lower in HCLE-SLURP1 compared with HCLE cells. Upon TGF-b treatment, E-cadherin expression decreased to a greater extent in HCLE compared with HCLE-SLURP1 cells. Confluent HCLE-SLURP1 cells retained E-cadherin in their cell membranes more efficiently than HCLE cells at 24h and 48h after TGF-b addition.
Conclusions :
Increased expression of TGFBR2, Smad4 and pSmad2/3 indicate higher canonical TGF-b signaling activity in Slurp1X-/- corneas. SLURP1 suppresses canonical TGF-b signaling in HCLE cells and protects E-cadherin from TGF-b induced disintegration, consistent with a role for SLURP1 in protecting the CE cells from TGF-b-induced EMT.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.